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Tetrahydrobiopterin and its functions B. Thöny Division of Clinical Chemistry and Biochemistry, University Children’s Hospital, Zurich, Switzerland This article is dedicated to my collaborator, Walter Leimbacher, who died on June 4, 2004 at the age of 58. Introduction Tetrahydrobiopterin (BH4) is essential for diverse processes and is ubiquitously present in all tissues of higher organisms. It is well established as essential cofactor for various enzyme activities to activate dioxygen, and for less defined functions at the cellular level. The latter function of BH4 seems to have a dual nature, depending on the concentration of the cofactor and the cell type, between proliferative activity and trigger for apoptosis. Since BH4 (and related tetrahydropterins) can form radicals, it can act as a generator or scavenger of reactive oxygen species in cells. More characteristics are arising based on recent observations, including chaperon function reported at least for the hepatic phenylalanine hydroxylase. The BH4 cofactor is endogenously synthesized and a (genetic) deficiency in the biosynthesis or regeneration leads to neurological abnormalities, including Dopa-responsive dystonia or severe monoamine neurotransmitter depletion. However, also under normal BH4 concentrations its availability becomes limiting in various pathological situations involving endothelial dysfunction, for instance, in diabetes or coronary heart diseases. From such developments it is expected that research on cofactor function will become of even broader interest in unraveling pathophysiological connections. The following overview is basically an extension of a previous review article on the biosynthesis, regeneration, and functions of the BH4 cofactor [1]. The main advances since the last review involve progress on the GTP cyclohydrolase I feedback regulatory protein (GFRP) with structural and functional aspects, on the postulation of alternative pathway steps for the biosynthesis of BH4 in peripheral organs based on the discovery of patients with sepiapterin reductase deficiency, on the function of BH4 cofactor as a chaperon, and on the generated mouse models for BH4 deficiency. The focus of this overview will again be on the structure and function of the mammalian enzymes responsible for the synthesis and regeneration of BH4. (A comprehensive review covering BH4 biology also in nonmammalian species can be found in [2]). Taken together, advances in the knowledge of cofactor function is not solely an academic exercise for biochemists and geneticists, but should be the basis for understanding the pathophysiology in order to eventually help patients with different defects involving directly or indirectly the BH4 cofactor. 495 Biosynthesis of BH4 In the following, the de novo biosynthesis of the BH4 cofactor carried out by the three classical enzymes is described. In addition, a hypothetical alternative way to circumvent the last enzymatic step by involving other reductases is presented (from compound 5 to 11 in scheme 1; see also below). Part of this alternative pathway is also called salvage pathway, which feeds BH4 from dihydrobiopterin via the NADPH-dependent dihydrofolate reductase. As will be discussed, and in contrast to our previous view, this alternative pathway seems to compensate for a genetic defect in the last biosynthetic step at least in peripheral tissues. Reaction mechanism of the de novo pathway BH4 biosynthesis proceeds in the de novo pathway in a Mg2+-, Zn2+-, and NADPHdependent reaction from GTP via the two intermediates, 7,8-dihydroneopterin triphosphate (H2NTP, compound 5) and 6-pyruvoyl-5,6,7,8-tetrahydropterin (PTP, compound 8), which have been isolated although they are rather unstable (Figure 1). The three enzymes GTP cyclohydrolase I (EC 3.5.4.16; GTPCH), 6-pyruvoyl-tetrahydropterin synthase (EC 4.6.1.10; PTPS), and sepiapterin reductase (EC 1.1.1.153; SR) are required and sufficient to carry out the proper stereospecific reaction to 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (compound 11). With the crystallographic structures, including the characteristics of the active centers of all three enzymes, the essential information for the interpretation of the reaction mechanism is available. Moreover, NMR studies on the reaction mechanisms of all three enzymes revealed the details of the hydrogen transfer process and the stereochemical course of the reactions [3]. The committing step is carried out by GTPCH, a homodecamer containing a single zinc ion in each subunit, consisting of a tightly associated dimer of two pentamers [4, 5]. GTPCH contains 10 equivalent active centers with 10-Å deep pockets. The interface of 3 subunits, two from one pentamer and one from the other forms an active site. This cavity is structurally stabilized by two loops (residues 109–113 and 150–153 in the E. coli enzyme) formed by hydrogen bond interactions and a salt bridge (Glu111–Arg153). An intramolecular disulfide bridge (Cys110–Cys181) is not essential for active site integrity as shown by Cys-mutant structures [6]. The atomic structure of this pocket is not only highly selective for GTP, but also provides residues for complete charge compensation in order to render obsolete Mg2+-assisted binding to the protein, as found in other nucleoside triphosphate-binding proteins. 496 O 1 4 HN 3 2 H2N PPPOH2C5‘ 1 N 5 9 8 HN 3 N N 2 O 4‘ H2N GTP 1‘ 3‘ O 11 7 6 4 1 N 4a 5 8 +NADPH - NADP+ H N N N H O H N N N H HN CHO N O 10 NH HN NH H2N O OH SR O OH OH +H2O -HCOOH 9 HN O GTPCH OH OH O 3 7 N H +H2O H2N PPPOH2C 6 5,6,7,8-Tetrahydrobiopterin 2‘ OH 2 OH H N HN N H2N H2N NH2 OH O N OH +NADPH - NADP+ - O - P O O P O O O OH O O HO O - 8 HN 3 2 - O P P O O O O H2N N O OH - O H2N OH 4 N 1 8 N - O P P O O O O 5 N H 1‘ 6 7 HN H2N 2‘ 1‘ 7 O N H N H O H N N N H 6 2‘ 3‘ OPPP OH HN H2N H N N PTPS O - -H2O 7 P O O O P O O O HO 2 6 -PPP OH HN3 8 NH O 5 N 5 NH2 HN H2N 1 O H N 6-Pyruvoyl-tetrahydropterin O 4 4 O OPPP OH OH OPPP OH 7,8-Dihydroneopterin triphosphate Scheme 1: Reaction mechanism for the de novo pathway for BH4 biosynthesis 497 498 Figure 1: Three-dimensional structures of BH4 metabolizing enzymes. Ribbon-type respresentations of the main-chain foldings of the proteins and enzymes involved in de novo BH4 biosynthesis and regenaration are shown: GTPCH, GTP cyclohydrolase I (homodecamer); GFRP, GTP cyclohydrolase feed-back regulatory protein (homopentamer); PTPS, 6-pyruvoyl tetrahydropterin synthase (homohexamer); SR, sepiapterin reductase (homodimer); PCD/DCoH, pterin-4a-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor-1α (homotetramer); DHPR, dihydropteridine reductase (homodimer). Substrates are shown in ball-and-stick representation with atoms in standard colors. On the right side the enzymes are shown rotated by 90º around the x-axis. The crystal structure coordinates used are GTPCH from E. coli (Protein Data Bank entry code 1GTP), GFRP from rat (1JG5), PTPS from rat liver (1GTQ, 1B66), SR from mouse (1SEP), PCD/DCoH from rat (1DCH, 1DCP), and DHPR from rat liver (1DHR). The figure was created using the programs MOLSCRIPT (Kraulis, 1991, J Appl Crystallogr 24:945), RENDER (Merrit and Murphy, 1994, Acta Crystallog Sct D Biol Chrystallogr D50:869), and/or PyMOL (http://pymol.sourceforge.net/). A catalytic mechanism was proposed based primarily on structural analysis obtained from E. coli GTPCH co-crystals with the dGTP analog and several active site mutants, and single turn-over experiments using a kinetically competent reaction intermediate [6, 7] (see Scheme 1). The purine hydrolysis reaction may be initiated by protonation of N-7 by a specific histidine residue (His179 in the E. coli enzyme). Attack of water at C-8 and suc- 499 ceeding opening of the imidazole ring result in a first intermediate, the 2-amino-5-formylamino-6-ribofuranoside triphosphate (compound 2). Protonation of the bridging O atom in the furanose ring and release of C-8 of GTP by another histidine residue (His112, E. coli) as formate yields the Schiff’s base intermediate 3. The subsequent Amadori rearrangement catalyzed by involving the γ-phosphate of GTP and a serine residue (Ser135, E. coli), and keto-enol tautomerization results in the intermediate compound 4. The last step is the ring expansion by closing between N-7 and C-2’, yielding the product H2NTP (compound 5). It is assumed that this last reaction step takes place rather at the protein surface or in solution after dissociation, as the spatial structure of the active site pocket does not favor the reaction to occur inside. The reaction from H2NTP (compound 5) to PTP (compound 8) is catalyzed by PTPS in a Zn2+- and Mg2+-dependent reaction without consuming an external reducing agent (Figure 1). This conversion involves a stereospecific reduction by an internal redox transfer between atoms N-5, C-6, and C-1’, oxidation of both side-chain hydroxyl groups, and an unusual triphosphate elimination at the C-2’-C-3’ bond in the side chain. Crystallographic analysis revealed that PTPS is composed of a pair of trimers arranged in a head-tohead fashion to form the functional hexamer [8]. The homohexamer contains six active sites that are located on the interface of three monomers, two subunits from one trimer and one subunit from the other trimer. The catalytic center and the reaction mechanism were studied by crystallographic and kinetic analysis of wild-type and mutant PTPS from the rat [9, 10]. In addition, the crystal structure of the inactive mutant Cys42Ala PTPS in complex with its natural substrate H2NTP was determined [10]. Each catalytic center harbors a Zn2+-metal binding site in a 12-Å deep cavity. The active site pocket with the specific pterin-anchoring Glu residue for salt-bridging plus two hydrogen-bonding amino acids appears to be similar to the equivalent sites in GTPCH, sepiapterin reductase, dihydroneopterin epimerase, and neopterin aldolase. The active site pocket contains in addition two catalytic motifs: a Zn2+-binding site and an intersubunit catalytic triad formed by a Cys, an Asp, and a His residue. The tetravalent co-ordination of the transition metal is accomplished via the Nε-atoms of three His residues and a fourth ligand provided by the pyruvoyl moiety of the H2NTP substrate. Unfortunately, neither the triphosphate nor the putative Mg2+ ion moieties could be defined in the electron density map. Zn2+ plays a crucial role in catalysis as it activates the protons of the substrate and stabilizes the intermediates (compounds 6 and 7). The proposed reaction mechanism is the following: Protonation of N-5 and abstraction of a proton from the C-1’ side-chain carbonyl atom lead to the N-5-C-6 double-bond reduction (compound 6). Stereospecific protonation of C-6 and oxidation of C-1’-OH to C-1’=O leads to compound 7. The last step is the abstraction of a proton from the C-2’ carbon of the carbonyl side-chain, followed by triphosphate elimination and tautomerization to yield PTP (compound 8). The Cys residue (Cys42 in the rat enzyme) of the catalytic triad appears to be the general base for stereospecific protein abstraction in both reaction steps. 500 The final step is the NADPH-dependent reduction of the two side-chain keto groups of PTP (compound 8) by SR. The overall structure of SR is a homodimer stabilized by a common four-helix bundle [11]. Each monomer contributes with two α-helices to the central dimerization domain and forms a separate complex composed of seven parallel β-sheets surrounded by α-helices. The C-terminal end of the β-sheets contains in close vicinity NADPH and the pterin binding site, the latter comprising a 15-Å deep pocket. The pterin substrate is anchored by the guanidino moiety of a specific Asp (Asp258 in the mouse SR; Asp258 is also the anchoring residue for N-acetyl serotonin inhibitor binding; see below). The C-1’ carbon of the pterin side-chain is in direct proximity to NADPH and a Tyr OH-group (Tyr171 in the mouse SR). This Tyr is the central active site residue for optimal proton transfer [12]. Based on kinetic, crystallographic, and NMR data, the initial step is the NADPH-dependent reduction at the side-chain C-1’-keto function leading to the formation of 1’-OH-2’-oxopropyl tetrahydropterin (compound 9) [3]. Internal rearrangement of the keto group via side-chain isomerization leads to the 1’-keto compound 6-lactoyl tetrahydropterin (compound 10). Intermediate compound 10 is then reduced to BH4 (compound 11) in a second NADPH-dependent reduction step. While the pterin substrate remains bound to the active site, the redox cofactor has to be renewed after the first reduction. It is thus assumed that NADP is exchanged at the opening located at the opposite side of the pterin-binding and entry pocket. Alternative routes for biosynthesis of BH4 Besides the involvement in the de novo biosynthesis of BH4, SR may also participate in the pterin salvage pathway by catalyzing the conversion of sepiapterin (Scheme 2, compound 17) to 7,8-dihydrobiopterin (compound 16) that is then transformed to BH4 by dihydrofolate reductase (DHFR; EC 1.5.1.3) [13]. Both reactions consume NADPH. Although SR is sufficient to complete the BH4 biosynthesis, a family of alternative NADPHdependent aldo-keto reductases, including carbonyl reductases (CR), aldose reductases (AR), and the 3α-hydroxysteroid dehydrogenase type 2 (AKR1C3) may participate in the diketo reduction of the carbonyl side-chain in vivo [14–16]. Moreover, based on the discovery of the autosomal-recessive deficiency for SR, which presents with neurotransmitter deficiency but without hyperphenylalaninemia (see also below), different routes for the final two-step reaction of BH4 biosynthesis involving these alternative reductases were proposed [16, 17] (Scheme 2). CR converts 6-pyruvoyl tetrahydropterin (compound 8) to both, the 2’-oxo-tetrahydropterin (compound 9) and the lactoyl tetrahydropterin (or 1’oxo-tetrahydropterin; compound 10); however, the rate of production is much more favorable for the 1’-oxo-tetrahydropterin intermediate. On the other hand, the 3α-hydroxysteroid dehydrogenase type 2 efficiently converts compounds 8 to 9. AR can convert 6pyruvoyl tetrahydropterin to 6-lactoyl tetrahydropterin (compound 10), or to 2’-oxotetrahydropterin (compound 9) to BH4. In summary, alternative routes for BH4 in the absence of SR involve in one pathway option via the 2’-oxo-tetrahydropterin the concerted action of 3α-hydroxysteroid dehydrogenase type 2 and AR, thereby consuming 2 501 NADPH. In the other pathway option via the 1’-oxo-tetrahydropterin intermediate, AR, CR, and dihydrofolate reductase are required. This second route involves consecutively the compounds 8, 10, 17, 16 to 11, and consumes 3 NADPH due to a non-enzymatic reduction step to sepiapterin (from compound 10 to 17). It was proposed that due to low expression or activity of dihydrofolate reductase and 3α-hydroxysteroid dehydrogenase type 2 in human brain, the biosynthesis from 6-pyruvoyl tetrahydropterin to BH4 in the absence of SR cannot be completed. This leads to central BH4 deficiency with accumulation of the unstable 1’-oxo-tetrahydropterin (compound 10) and its degradation products, detected as ‘abnormal’ pterin metabolites, i.e. compound 16. 8 O HN 3 2 AR/CR H2N +NADPH - NADP+ 17 O H2N O non-enzymatic N HN N N H OH H2N N N 7 N H DHFR OH 7,8-Dihydrobiopterin +NADPH - NADP+ 2‘ 1‘ O SR/AKR1C3/CR +NADPH - NADP+ 6-Pyruvoyl-tetrahydropterin 9 O O H N N N H HN N SR OH N H SR OH N H 8 1 O 6 6-Lactoyl tetrahydropterin N HN H2N H N +NADPH - NADP+ O 16 O HN Sepiapterin SR/CR 10 H N 5 4 H2N OH O 1‘-Hydroxy-2‘-oxopropyl tetrahydropterin +NADPH - NA DP+ +NADPH - NADP+ 11 O HN3 2 H2N 4 1 N 4a H N 5 OH 8 7 N H AR 6 OH 5,6,7,8-Tetrahydrobiopterin Scheme 2: Reaction mechanism for alternative routes for BH4 biosynthesis Enzyme structures GTPCH and GFRP Structural crystallographic data are available for the recombinant GTPCH from the human and the E. coli enzymes [4, 5,18,19], for the rat GFRP [20], and for the GTPCH-GFRP cocomplex from rat [19]. The homodecameric GTPCH is composed of two dimers of pentamers (Figure 1). Regarding the E. coli enzyme, each subunit contains 221 amino acids and folds into an α+β structure with a predominantly helical N-terminus. The N-terminal antiparallel helix pair α2/α3 is remote from the rest of the molecule. The compact C-terminal body of the monomer (residues 95–217) is formed by a central four-stranded antiparallel β-sheet (β-1 to β-4) that is flanked on both sides by α-helices (α4, α5, and α6) (Figure 2A). The N-terminal helix α1 lies on top of helices α4 and α5. The association of 502 two GTPCH monomers to dimers is driven by the formation of a four-helix bundle by helices α2 and α3. Five monomers interact along their β-sheets to form a 20-stranded antiparallel β-barrel with a diameter of 35 Å. The decamer is formed by a face-to-face association of two pentamers whereby the antiparallel helix pair α2/α3 of one monomer is intertwined with that of another monomer. The decamer has a toroidal shape with an approximate height of 65 Å and a diameter of 100 Å. It encloses a cavity of 30 Å x 30 Å x 15 Å that is accessible through the pores formed by the five helix bundles in the center of the pentamers, but has no opening at the decamer equator. The active site is located at the interface of three subunits, two from one pentamer and one from the other. There are thus ten equivalent active sites per functional unit. The GTPCH contains a zinc ion bound to the active site, i.e. bound to Cys-110, His-113, and Cys-181 in the bacterial enzyme (complexed by Cys-141, His-143, and Cys-212 in the human GTPCH). Sequence information of a number of GTPCHs from diverse organisms is available (Figure 2A). High overall sequence similarity is found among the mammalian enzymes (approximately 90% identity). A comparison of all sequences reveals that mainly the Cterminal sequence is evolutionarily conserved; i.e. the C-terminal 120 residues between the human and the E. coli enzymes are 60% identical. Moreover, almost all residues participating in pterin binding and/or catalysis appear to be conserved. This high sequence homology suggests that the tertiary and quaternary structures of GTPCH are most probably very similar, an assumption that was confirmed by solving the structure of the rat [19] and human enzymes, the latter with a deletion of amino acid residues 2–41 [5]. The high diversity of the N-termini between the mammalian and the non-vertebrate enzyme, most notably the N-terminal extension in the Drosophila sequence, may be due to different regulating functions such as, for instance, docking sites for the GFRP (see also below). In the 360 kDa GTPCH-GFRP complex, the decameric GTPCH is sandwiched by two GFRP homopentamers. The model for the rat complex contains 10 GTPCH units (residues 48–241) and 10 GFRP subunits (residues 1–84), 10 phenylalanine molecules, and 10 zinc ions (Figure 1). Each monomer is folded into an α/β-structure with a sixstranded antiparallel β-sheet and two α-helices, arranged onto a ββαββαββ topology. The monomers form a propeller-like pentameric-disc with an overall dimension of 50 Å x 50 Å x 20 Å for this complex. The contacts between GFRP and GTPCH are, besides salt bridging, primarily due to van der Waals interactions, as the concave side of GFRP pentamer exhibits neutral to negative surface potential, whereas the contact surface of the GTPCH is mainly positively charged. The phenylalanine binding sites are located at the interface between GFRP and GTPCH, and phenylalanine binding enhances the association of these proteins. The complex structure suggests that phenylalanine-induced GTPCHGFRP complex formation enhances GTPCH activity by locking the enzyme in the active site. 503 504 Figure 2: Amino acid sequence alignments of BH4 synthesizing enzymes. Alignments were generated using the Clustal W program [216]. Identical residues and residues with functional similarities (V/L/I, R/K, M/L, D/E) are highlighted with shaded boxes. Residues involved in catalysis and/or substrate binding are marked with a triangle. The identified phosphorylation site in PTPS (Serine 19 in the human sequence) is marked with a star. In case of SR, residues involved in cofactor binding are additionally A) GTPCH are marked. The accession numbers for the amino acid sequences for (A NP_000152 (human), AAA37757 (mouse), AAA41299 (rat), NP_523801 (Drosophila), E. coli ); for (B B) PTPS are CAA87397 (yeast), Q94465 (Dictyostelium), CAA45365 (E C) Q03393 (human), AAD15827 (mouse), P27213 (rat), P48611 (Drosophila); and for (C SR are P35270 (human), Q64105 (mouse), P18297 (rat), AAC60297 (Fugu), and D) GFRP are NP_005249 (human), NP_796131 (mouse), AAD12760 (Drosophila). (D NP_598279 (rat). 505 Sequence information is available for the human, rat, and mouse GFRP, which share 94% sequence homology with conservation of the secondary structural elements (Figure 2D; GTPCH is not regulated by GFRP in bacteria which do therefore not contain the corresponding protein). PTPS Only the recombinant rat liver enzyme could be crystallized, which yielded interpretable diffraction data (residues 7–144) [9]. Each subunit folds into a compact, single-domain α+β structure (Figure 1). The monomer consists of a sequential, four-stranded, antiparallel β-sheet (β-1 to β-4) with a 25-residue, helix-containing (αA) insertion between β-1 and β-2 at the bottom of the molecule (Figure 2B). Strands 3 and 4 are connected via an αhelical turn. A segment between strands 2 and 3 forms a pair of antiparallel helices, αB and αC, layered on one side of the four β-sheets. Three monomers assemble into a trimer, forming a 12-stranded antiparallel β-barrel structure surrounded by a ring of α-helices. Crystallographic and experimental data revealed that the mammalian PTPS is homohexameric and dissociates into trimers. Two trimers arrange in a head-to-head fashion to form a barrel, the functional PTPS hexamer with an overall shape of 60 Å x 60 Å x 60 Å. Due to the relatively tilted order of the β-sheet, the pore in the trimer is conically shaped with a 6–12 Å diameter. In the hexameric enzyme, the pore has a smaller opening towards the trimer interface, and opens up also equatorially to the hexamer surroundings. The inside of the barrel accumulates a cluster of basic and aromatic residues stretching radially into the pore. Each subunit of the homohexamer contains one putative active site that is located at the interface of three monomers, two subunits from one trimer and one subunit from the other trimer. Each active site harbors three histidine residues (His23, His48, and His50 in the rat enzyme), with the Ne-atoms coordinating the binding of the Zn2+-metal. In the unliganded state a water molecule is bound as the fourth ligand. In a substrate complex structure the C1’ and C2’ side-chain hydrolysis of the dihydroneopterin ligand displaces the bound water, yielding a pentavalent co-ordination of the transition metal [10]. Complete PTPS sequences from several mammals and from Drosophila are available, while partial amino acid sequences from salmon are also known (Figure 2B). With the exception of the C-terminal extension in the fly enzyme, the overall amino acid identity is around 80%. All residues involved in substrate binding and catalysis are conserved among these sequences. The high degree of subunit conservation between the species implies that all these PTPS form a homohexameric structure. SR The 1.25 Å-crystal structure of the mouse SR in complex with NADP has been solved [11]. The 261 amino acids of the monomer fold into a single domain α/β-structure. A sev506 en-stranded parallel β-sheet, βA–βG, in the center of the molecule is sandwiched by two arrays of three a-helices (αC, αB, αG and αD, αE, αF) (Figure 2C). The association of two monomers to the active homodimeric SR leads to the formation of a four-helix bundle (Figure 1; helices αE and αF of each monomer). Due to the two-folded crystallographic symmetry of the homodimeric molecule, the parallel β-sheets in monomer A is in an antiparallel orientation relative to the β-sheet of monomer B enclosing an angle of 90°. The overall dimensions of the SR dimer are 40 Å x 50 Å x 80 Å. The two substrate pockets bind sepiapterin (or 6-pyruvoyl-tetrahydropterin; compound 8 in Figure 1), and the cofactor NADP/NADPH from opposite sides to the enzyme. Amino acid sequence comparison for the available SRs revealed a high degree of similarity among the mammalian enzymes (around 90%), as well as a high degree of conservation compared to the fish and fly enzymes (Figure 2C). It is thus assumed that the tertiary and quaternary structures of SR are conserved throughout these species. From both amino acid sequence and three-dimensional structure, SR can be assigned to the family of short-chain dehydrogenases/reductases. They all use NAD(H) or NADP(H) as the cofactor and contain a strictly conserved Tyr-X-X-X-Lys sequence motif (Tyr171 and Lys175 in the mouse SR sequence). Comparison of the three biosynthetic enzymes There is no sequence identity among the three BH4-biosynthetic enzymes. However, a comparison of polypeptide secondary structure revealed that the C-terminal domain of GTPCH shows a similar subunit fold with PTPS, and also with neopterin aldolase, neopterin epimerase, and uroate oxidase, whereas the overall structure of SR is entirely different [21, 22]. The tertiary structure of GTPCH and PTPS revealed for both enzymes a central pore for which the function is still unknown; at least it seems not to be directly involved in catalysis and substrate binding. A speculative but attractive role for these cavities might be ‘channeling’ of the unstable pterin intermediates from one enzyme to the other (pterin compounds 5 and 8). A potential substrate channeling would imply that the biosynthetic enzymes must interact and thus form some type of a super-complex. Such physical interaction has not yet been observed. In terms of complex formation with their natural substrates, all three enzymes show the same active site architecture. Furthermore, this pterin-binding motif unit is quite similar to the GTP-binding motif in small GTP-binding proteins [11]. 507 Genes encoding the biosynthetic enzymes The corresponding genes for all four proteins, including GFRP, are known from human and mouse, in some instances also from other organisms. An overview of gene names, chromosomal location, and exon content for the human genes is presented in Table 1. Enzyme (EC number) Gene Chromosomal location No. of exons No. of amino acid residues Size kDa x no. of subunits 27.9 x 10 GTPCH (3.5.4.16) GCH1 14q21.1.-22.2 6 250 GFRP GCHFR 15q15 3 84 9.7 x 5 x 2 PTPS (4.6.1.10) PTS 11q22.3-23.3 6 145 16.4 x 6 SR (1.1.1.153) SPR 2p13 3 261 28.0 x 2 PCD/DCoH (4.2.1.96) PCBD 10q22 4 104 11.9 x 4 DHPR (1.6.99.7) QDPR 4p15.3 7 244 25.8 x 2 Table 1: Human BH4-metabolizing enzymes. For references see text or the NCBI database (www.ncbi.nlm.nih.gov). Note that PCD/DCoH is homotetrameric (α4) only as a carbinolamine dehydratase, whereas in complex with HNF-1α it is heteroterameric (α2β2). Furthermore, a second copy for PCD/DCoH, DCoH2, was identified (for details see text). GFRP forms two pentameric complexes with the decameric GTPCH. GCH1 Human and mouse GTPCH is encoded by a single copy gene, GCH1, and is composed of 6 exons spanning about 30 kb [23]. The location of the 5 introns in mouse and human genes is at identical sites. In the fruit fly Drosophila, GTPCH is encoded by the Punch locus. There, three out of four introns have identical positions (introns 2–4 [24]), whereas the single intron present in the Dictyostelium discoideum gene is inserted at a different site [25]. The yeast gene has no intronic sequences [26]. Although complete cDNAs for the GTPCH were isolated from many diverse organisms (see for instance [27]), information on gene organization is mostly missing. Alternative splicing was observed for human exons 5 and 6, and Drosophila exon 1. Up to four types of GTPCH-cDNAs with different 3’ ends were isolated from human liver or cell lines [28, 29]. Type 1 cDNA with 250 codons has the longest coding region and the greatest similarity to that reported from rat and mouse [30, 31]. Furthermore, the full-length type 1 GTPCH-cDNA was isolated from a pheochromocytoma cDNA library [32]. Alternative usage of the splice acceptor site within exon 6 generates the shorter type 2 mRNA [23, 33]. Type 3 mRNA contains, besides exon 1–5, an extension of exon 5 due to non-usage of the splice sites from ‘intron 5’. The type 4 mRNA variant is spliced within exon 6 without affecting the reading frame. Putative proteins derived from type 2 and 3 mRNAs miss the C-terminal 40 amino acids- 508 containing residues involved in pterin binding and catalysis, and are highly conserved compared to the E. coli enzyme (see Figure 2A). In agreement with this is the observation that individual expression of recombinant proteins from these cDNAs in bacterial cells yielded GTPCH activity only with the type 1 cDNA. It was further demonstrated that the type 2 mRNA exerts a dominant-negative effect on the wild-type cDNA, similar to the effect of some GCH mutants [34], and may thus contribute to the regulation of BH4 production in specific cells [33]. Drosophila contains two 5’ splice variants for the GTPCH mRNA. These alternative exons confer distinct N-terminal domains to each predicted protein and cannot be aligned with the N-termini of any other GTPCH. It was speculated that different GTPCH isoforms in Drosophila and in higher eukaryotes might have specific subcellular and/or tissue distribution, and that alternative isoforms could associate and respond to different regulatory or modifying proteins. So far there is no experimental evidence for such regulatory diversity. However, it was observed that two different complex forms of GTPCH activities exist in human liver upon separation by molecular mass (400 kDa and 600 kDa). In this case both forms contained the same protein subunit as component with the identical mass as judged by SDS-PAGE analysis [35]. The transcription start sites and analysis for the upstream promoter sequences from the human, rat, and mouse GCH1 genes are available. Functional cis-acting elements for basal, cAMP, and RNF4/NF-Y dependent expression, including a CRE-Sp1-CCAAT-box were described within the first 150 bp upstream of the cap sites in these promoters [36–41]. However, the sequences neither revealed typical sites known to be involved in for instance interferon-γ signal transduction, nor was any response observed upon interferon-γ treatment of transfected human cells with corresponding reporter constructs. Since it is well established that GTPCH gene expression can be induced by interferon-γ (and other compounds; see below) in various rodent and human cells (see below), further studies are necessary to characterize the mammalian GCH1 promoter. PTS Organization of the human and mouse PTS genes is highly conserved. They are both spanning a region of 6–7 kb of genomic DNA and contain six exons with their introns at identical positions [42–44]. Originally, a splicing polymorphism that occurred in at least some human cell types leading to skipping of the 23 bp exon 3, and thus aberrant protein expression was detected in normal controls (and in patients). Nonetheless, all cells and tissues investigated appeared to have expressed the same human PTPS-mRNA. However, a more detailed study revealed that the amount of exon-3-containing mRNA was correlated closely to PTPS activity, concluding that exon 3 skipping during transcript splicing is a major cause or regulator of the low PTPS protein expression in different human cell types (see also below) [45]. The human but not the mouse genome contains in addition a retropseudogene, PTS-P1, located on chromosome 9q13. In this retropseudogene, the 5’ 25 codons and an internal fragment of 23 bp corresponding to exon 509 3 (codons 54–61) are entirely absent. The overall similarity to the 3’ portion of the PTPScDNA is 74%. The Drosophila purple gene produces two PTPS-mRNAs: a head specific one with three introns expressed from a proximal promoter and a constitutive mRNA with an additional intron in the 5’ non-translated region expressed from a distal promoter [46]. The reading frame encoding PTPS from both mRNAs is the same. When compared to the human and mouse intron-exon organization, the first two introns in the Drosophila genecoding sequence have identical positions, and the third Drosophila intron is located two codons upstream of the corresponding human intron 5 position. No information is thus far available on transcriptional start sites and corresponding promoter analyses for the mammalian PTS genes. SPR Genomic organizations of the mouse and human SPR genes encoding SR are very similar [47, 48]. They both span a region of 4–5 kb and the reading frames are split into 3 exons. No alternative splice variants have been observed. Only the mouse harbors a genomic pseudogene (Sprp) containing exons 1 and 2 plus the intervening and partial flanking sequences for these two exons with an overall similarity to the functional SPR gene of 82%. The single gene for SR in Drosophila contains no introns and encodes a single transcript of 1.4 kb that is present in heads and bodies of adults [49]. In all three species, the transcriptional start sites (+1) were determined for the SPR genes [47, 48]. No TATA-like sequences and no CAAT-box motifs were found in the upstream vicinities of the +1 sites. Furthermore, for the mouse SPR fusion studies with a reporter gene were conducted revealing that the promoter contains a sequence between –83 and –51 upstream of the transcriptional start site that is essential for expression. GCHFR The cDNAs for the rat and human GFRP proteins were described [50, 51], but cDNAs and/or genes structures from other organisms are also available on the NCBI database (see also Figure 2D). The corresponding human GCHFR gene, which is located on chromosome 15q15, contains 3 exons and spans a region of roughly 4 kb (accession number U78190). Although promoter elements have not yet been described, transcription analyses in murine or human tissues by Northern blots (or RT-PCR) revealed the presence of up to three different GCHFR-specific transcripts in all samples investigated, including brain [51, 52]. 510 CpG abundance An increased abundance of the dinucleotide CpG in a DNA sequence of at least 200 bp spanning exon 1 and the transcriptional start site in comparison with the residual DNA sequence of a gene are known to be typical for housekeeping genes (C+G content >50%, frequency of observed vs. expected CpG =0.6; [53]). Such a CpG content analysis with the human and mouse BH4-biosynthetic genes revealed, as expected, that GCH1 is a regulated gene, whereas PTS and SPR are predicted to be constitutively expressed (not known for GCHFR; conducted with the CpGPlot program by Gardiner-Garden and Frommer; [1, 54]). However, more detailed analyses are required to better define the promoters for the mammalian BH4-biosynthetic genes. Regulation of enzyme expression and activity Regulation of BH4 biosynthesis appears to be complex, and an integrated picture of the signal transduction and control pathways does not yet exist. Depending on the cell or tissue type, all enzymes are thought to be constitutively expressed, such as in the liver or in some brain regions. In other tissues, enzyme expression can be induced or is completely absent. Expression of GTPCH at least is inducible, and PTPS activity can be elevated to some extent. Moreover, post-translational modification(s) of all three biosynthetic enzymes and regulation of GTPCH by the GFRP may modulate enzymatic activities. A detailed review on the regulation of BH4-biosynthetic enzymes can also be found in [2]. GTPCH The committing step for BH4 biosynthesis is the major controlling point for cofactor biosynthesis. GTPCH activity can be regulated at the transcriptional and post-translational levels and by the GTPCH-interacting protein GFRP that modulates enzyme activity. In tissues like liver and many brain regions that synthesize monoamine neurotransmitters, GTPCH is constitutively present [55], whereas expression is inducible in virtually all other cell types or organs [2]. The regulation on the transcriptional level by various immune stimuli such as cytokines, phytohemagglutinin, and endotoxin (lipopolysaccharide) in a cell- and tissue-specific mode is probably predominant. Cytokines such as interferon-γ, tumor necrosis factor-α, stem-cell factor (or kit ligand), interleukin-1β, glial cell linederived neurotrophic factor (GDNF), or a specific combination of these, induce GTPCH gene expression in vitro and/or in vivo in various human and rodent cells, including T-lymphocytes, macrophages, monocytes, fibroblasts, endothelial cells, smooth-muscle cells, bone marrow-derived mast cells, mesangial cells, glioma cells, rat lungs, and locus coeruoleus in mouse brain (for review see [2]; [56–59]). Some of these stimulatory agents, for instance lectin, may act in an indirect way by first triggering cytokine release from Tlymphocytes, which then stimulates GTPCH activities in other blood cells. In humans, a 511 biochemical consequence of immunostimulation is the excretion of both neopterin and 7,8-dihydroneopterin by activated macrophages and consequently accumulation in plasma and urine. A physiological function for these compounds has not been established. Lipopolysaccharide-treated rats show de novo expression and increased enzyme activity of GTPCH in brain, liver, spleen, and adrenal gland. In cultured dopamine neurons of the hypothalamus and mesencephalon, or in the human neuroblastoma cell line SK-N-BE, but also in other cell types, cAMP and depolarization of membrane potential were found to stimulate GTPCH-mRNA expression [39, 60]. Increased levels of GTPCH mRNA were also observed in peripheral and central neurons upon treatment with the catecholaminedepleting drug reserpine [61], and in brain catecholaminergic regions with addition of estrogen [62], or by GDNF in primary dopaminergic neurons [59]. Furthermore, stimulation of GTPCH-mRNA expression was also reported upon phenylalanine treatment of HUVEC cells [52], and arginine or hydrogen peroxide (H2O2) in endothelial cells [63, 64]. On the other hand, anti-inflammatory cytokines, for instance IL-4, IL-10 and TGF-β, down-regulate GTPCH expression [2, 63]. Glucocorticoids or cyclosporin A were reported to down-regulate GTPCH gene expression, but also to have the opposite effect, i.e. to increase mRNA expression and enzyme activity (thereby decreasing the iNOS) [57, 65]. Opposing effects of glucocorticoids (and potentially other compounds) may depend on co-treatment with cytokines, and/or cell types [66] (for more detailed overiews see [63, 67]). Post-translational processing of GTPCH involves cleavage of the N-terminal 11 amino acids for at least the rat liver enzyme and protein phosphorylation [30, 68–70]. Whereas a regulatory effect for the N-terminal processing is not known, phosphorylation modulates enzyme activity: (1) Protein kinase C stimulators like phorbol ester, platelet-derived growth factor, and angiotensin II or specific inhibitors caused an increase or reduction, respectively, of GTPCH phosphorylation. (2) Concomitantly, phosphorylation coincides with elevated enzyme activity and an increase of cellular BH4 levels. (3) In vitro phosphorylation with purified enzymes demonstrated that GTPCH is modified by casein kinase II and/or protein kinase C. The primary amino acid sequence of GTPCH reveals several conserved sites for potential phosphorylation by casein kinase II. However, only one serine residue (Ser167 in the rat and mouse sequences), which is conserved between the human, rat, mouse and Drosophila enzymes, was proposed to be a potential target site for protein kinase C. Further effectors for GTPCH enzymatic regulation are its substrate GTP, the pathway-end product BH4 (and other reduced pteridines), phenylalanine, and calcium ions. BH4 as feed-back inhibitor and phenylalanine as feed-forward stimulator modulate enzymatic activity, with different affinities of these effectors, via two homopentameric GFRP that form a sandwich with the homodecameric GTPCH [50, 71–74]. A physiological consequence of GFRP action is the high plasma BH4 concentrations observed in patients with hyperphenylalaninemia caused by PAH deficiency [71]. Each GFRP has a cavity at the interface with GTPCH that binds phenylalanine and enhances the association of the GTPCH-GRFP 512 complex. Furthermore, this phenylalanine locks the enzyme in the active state [19, 74]. GFRP-mediated end-product inhibition by BH4 can be reverted to an active form by phenylalanine; however, the binding site for BH4 and its mechanism for GFRP-mediated feed-back inhibition are not clear. A functional high-affinity, EF-hand-like calcium-binding site was reported specifically for the eukaryotic GTPCH. It was speculated that mutant GTPCH with loss of calcium ion binding capacity might be a primary cause for doparesponsive dystonia (see below), however, the role for calcium binding remains unclear [75]. GFRP-mRNA studies by Northern blot analysis and in situ hybridization revealed that the expression pattern in rat tissues correlates with that of GTPCH, i.e. GFRP is expressed in peripheral organs like liver and heart, and also in the brain [50, 76]. Proinflammatory stimuli such as bacterial lipopolysaccharide or interferon-γ stimulate expression and activity of GTPCH, while down-regulating expression of GFRP, as shown in human cell lines or in vivo with rats. Moreover, down-regulation of GFRP-mRNA occurred also in presence of phenylalanine, thus rendering BH4 biosynthesis independent of metabolic control by phenylalanine [51, 52]. Furthermore, inhibition of GTPCH activity by 2,4-diamino-6hydroxypyrimidine (DAHP) was shown using recombinant proteins to be mediated by GFRP binding to its cognate GTPCH enzyme. Thus 2,4-diamino-6-hydroxypyrimidine is not competing with GTP at the substrate binding site of GTPCH [77]. PTPS The PTPS-mRNA is considered to be constitutively expressed but the protein or its activity is not ubiquitously present, at least not in higher animals; moreover, PTPS expression is strictly regulated in some cell types by an unknown mechanism at the transcriptional level (see below). Following immunostimulatory induction of GTPCH by cytokines, PTPS becomes the rate-limiting step in BH4 biosynthesis, at least in man, where PTPS activity is much lower than in rodents [78, 79]. Whereas GTPCH activity can be stimulated up to 100-fold in cytokine treated cells, PTPS activity remained unaffected in some experiments but was stimulated in others. In any case, PTPS activity was reported to be maximally elevated by a factor of 2 to 4 [45, 80–84]. Elevation of PTPS-mRNA (and GTPCH-mRNA; see above) by a factor of 3-4 was observed in rat adrenal glands following reserpine treatment [85]. A regulatory mechanism for PTPS expression at the mRNA (or pre-mRNA) level was observed for various human cell types, including blood-derived cells, fibroblasts, endothelial cells, and other cell lines [45]. Although normal levels of PTPS mRNA could be detected in all cells, PTPS protein presence and enzymatic activity were barely detectable in some cells. A detailed study of PTPS-mRNA revealed the presence of two types of PTPS transcripts: the normal functional mRNA and a species lacking the 23 bp-exon 3, which results in a premature stop codon. Furthermore, the ratio of exon-3-containing to exon-3-lacking PTPS-mRNA closely correlated to PTPS activity, and from this it was concluded that exon 3 skipping is a major cause of the low PTPS protein expression in these 513 cells. Furthermore, mRNA species lacking exon 3 were detected also in human brain cDNA libraries and in a PTPS-deficient patient [42, 43, 86]. The significance of such a post-transcriptional event as potential regulatory mechanism controlling the expression level of PTPS also in other cells remains to be clarified. O 11 HN H2N N H N N H 12 OH OH PAH OH O H N O +O2 N H2N N OH OH N H 5,6,7,8-Tetrahydrobiopterin Phe, Tyr,Trp +NADH - NAD + DHPR O 14 H2N OH -H2O N N N PAH N H OH PCD/DCoH 13 O OHH N N N H N H2N q-Dihydrobiopterin Tyr, L-Dopa, 5-OH-Trp OH OH Tetrahydrobiopterin4a-carbinolamine non-enzymatic O 16 HN H2N OH N N N H O 15 HN OH 7,8-Dihydrobiopterin H2N N H N OH N OH Primapterin (7-dihydrobiopterin) Scheme 3: Reaction mechanism for the BH4-regenerative pathway With regard to post-translational modifications, PTPS isolated from rat liver was found to be N-terminally processed by having the first four amino acids removed [87]. Furthermore, human PTPS was also shown to be subject to regulatory phosphorylation at Ser19, whereby the cyclic-GMP-dependent protein kinase type II seemed to be responsible for 514 the phosphoserine modification [88]. The molecular basis for the at least three-fold stimulation of the phosphorylated PTPS versus the non-modified protein when tested in COS1 cells is not yet understood. Nevertheless, phosphoserine modification appears to be essential as a phosphorylation-deficient mutant of PTPS was identified from a patient with a defect in BH4 biosynthesis [89]. It was speculated that in cultured dopamine neurons, where BH4 biosynthesis can be stimulated by cAMP, the observed short-term increase in BH4 levels may be attributed to cAMP-dependent phosphorylation of PTPS [85]. SR SR seems to be ubiquitously expressed, as it was found in all mammalian tissues, and its expression remains unaffected by cytokine treatment. A recent examination of the expression pattern in brain revealed that SR is expressed in all regions, in contrast to GTPCH or PTPS which are basically localized to monoamine neurons [55, 90] (A. Résibois and Thöny B, in preparation). Several alternative aldose-ketose reductases can substitute for SR (see below). Alternatively, SR may also have other functions as an aldose-ketose reductase besides being involved in BH4 biosynthesis (for instance detoxification [91]). Biochemical evidence for alternative reductases comes from the AKR family of enzymes that commit the last biosynthetic steps from 6-pyruvoyl-tetrahydrobiopterin to BH4 (compounds 8 to 11 in Scheme 2) [16]. The discovery of patients with defective SR who present with monoamine neurotransmitter deficiency but without hyperphenylalaninemia is probably the most compelling proof for the in vivo potential of alternative aldose-ketose reductases replacing SR in the BH4 biosynthesis [17, 92]. From N-terminal sequence analysis of SR purified from rat erythrocytes it is known that the protein begins with an N-acetyl-methionyl residue [93]. Furthermore, SR was reported to be phosphorylated in vitro by calmodulin-dependent protein kinase II and protein kinase C at one or several sites [12, 94]. Phosphorylation did not modify the kinetic properties of the purified rat and human enzymes, but it became more sensitive to calciumactivated proteases, a situation reminiscent of tyrosine hydroxylase [95]. Localization of biosynthetic enzymes As mentioned above, immunohistochemical staining with anti-GTPCH and anti-PTPS antibodies in rat brain revealed that the expression pattern is highly specific and co-localization was generally found with aromatic amino acid hydroxylases [55] (and A. Résibois and Thöny B, in preparation). In contrast, SR was detected in all brain regions examined, pointing towards additional functions as reductase besides being involved in BH4 biosynthesis [90]. In addition, studies with all 3 BH4-biosynthetic enzymes, GTPCH, PTPS, and SR, revealed some prominent nuclear localization in specific but various cell types, including neurons [96]. 515 Regeneration of BH4 Regeneration of BH4 is an essential part of the phenylalanine hydroxylating system (see also “Cofactor functions”). During the catalytic event of aromatic amino acid hydroxylases, molecular oxygen is transferred to the corresponding amino acid and BH4 is oxidized to BH4-4a-carbinolamine (Scheme 3) [97, 98]. Two enzymes are involved in its subsequent dehydratation and reduction to BH4: pterin-4a-carbinolamine dehydratase (EC 4.2.1.96; PCD) and dihydropteridine reductase (EC 1.6.99.7; DHPR). Enzymatic recycling of BH4 is essential for phenylalanine metabolism (1) to ensure a continuous supply of reduced cofactor, and (2) to prevent accumulation of harmful metabolites produced by rearrangement of BH4-4a-carbinolamine. The primary structure of PCD is identical with a protein of the cell nucleus that has transcription function, named dimerization cofactor (DCoH) of hepatocyte nuclear factor 1α (HNF-1α), recently reported to have general transcriptional function [99–101]. In the following, PCD will be designated as the dual-function protein PCD/DCoH. Furthermore, a functional homologue, DCoH2, was identified, which partially complements PCD/DCoH mutants in mice and man [102,103]. Reaction mechanism of the regenerative pathway The dehydratation of BH4-4a-carbinolamine (compound 13), the first product of the reaction of aromatic amino acid hydroxylases (Scheme 3), is catalyzed by the enzyme PCD/DCoH. The human cytoplasmic PCD/DCoH, whose sequence is identical to that of the rat protein, is a homotetramer with a molecular mass of 11.9 kDa per subunit (Figure 3A) [100,101]. Using chemically synthesized pterin-4a-carbinolamine it has been demonstrated that the enzyme shows little sensitivity to the structure or configuration of the 6substituent of its substrate, and to the 4a(R)-and 4a(S)-hydroxy stereoisomers [104]. Obviously, the binding pocket has a relatively high degree of flexibility and might not be designed to recognize only BH4-4a-carbinolamine. X-ray crystal structures of the tetrameric enzyme complexed with the product analogue 7,8-dihydrobiopterin (compound 16) revealed four active sites harboring three essential and conserved histidines (His61, His62, His79 in the human or rat enzyme; see Figure 3A; [105]). Detailed enzymatic studies on the stereospecificity and catalytic function revealed a dehydratation mechanism in which the three histidines in PCD/DCoH are crucial for activity [106–108]. A His61→Ala/His62→Ala double mutant was fully inactivated and showed a significantly increased dissociation constant of quinonoid 6,6-dimethyl-7,8-dihydropterin. Moreover, His61 and His79 act as general acid catalysts for the stereospecific elimination of the 4a(R)- and 4a(S)-hydroxyl groups, respectively (see Figure 3A). The role of His62 is primarily to bind substrate, with an additional component of base catalysis [107]. The quinonoid dihydrobiopterin (compound 14) product is a strong inhibitor of PCD/DCoH with a KI-value of about one half of its respective Km-value, and no inhibition was observed with 7,8-dihydrobiopterin (compound 16) [104]. Furthermore, PAH is not inhibited by its cofactor product, BH4-4a-carbinolamine, but by primapterin (compound 15). In the 516 absence of PCD/DCoH, dehydratation of BH4-4a-carbinolamine occurs also non-enzymatically, but at a rate that is, at least in the liver, insufficient to maintain BH4 in the reduced state [109]. As a consequence, liver PCD/DCoH deficiency in man causes BH44a-carbinolamine to be rearranged via a spiro structure intermediate to dihydroprimapterin (7-substituted dihydrobiopterin; compound 15) that is excreted in the urine [110,111]. Figure 3: Amino acid sequence alignments of BH4 regenerating enzymes. Alignments were generated using the Clustal W program [216]. Identical residues and residues with functional similarities (V/L/I, R/K, M/L, D/E) are highlighted with shaded boxes. Residues in PCD/DCoH involved in catalysis and/or substrate binding are marked with a triangle. For DHPR, the potential active site pocket residues are marked A) with a triangle. The accession numbers for the amino acid sequences for (A PCD/DCoH are P80095 (human), A47189 (rat), P61458 (mouse), AA06395 (chicken), AAC25196 (Drosophila); BAA17842 (cyanobacteria), AAC06420 (Aquifex), P43335 Pseudomonas aeruginosa ), DCoH2 Q9H0N5 (human) for (B B) DHPR are P09417 (P (human), P11348 (rat). 517 The final conversion of quinonoid dihydrobiopterin to BH4 is carried out by the dimeric DHPR (Scheme 3). Although the crystallographic structure of the DHPR-NADH binary complex was solved, the location of the active sites is not known from these studies. Nevertheless, an active site pocket involving the Tyr-X-X-X-Lys motiv (Tyr150 in the human DHPR), typical for short chain dehydrogenases, was proposed to participate in proton donation. Following the classical mechanisms of dehydratation, one molecule of water is released and the product quinonoid dihydrobiopterin is reduced back to BH4 in a NADHdependent reaction. This final reaction of the regeneration pathway involves direct hydride transfer from the reduced nicotinamide ring to the quinonoid dihydrobiopterin by DHPR. This reaction is supported by the proposed enzyme mechanism of NAD(P)H-dependent reductases and by the lack of detectable prosthetic groups such as flavin or metal ions [112]. The hydride transfer occurs from the B-face of NADH with transfer of the pro-S hydrogen. Enzyme structures PCD/DCoH The crystal structure of cytoplasmic PCD/DCoH from human and rat liver has been solved [113,114]. The single domain monomer of 103 amino acids comprises three α-helices packed against one side of a four-stranded, antiparallel β-sheet. The functional enzyme is a homo-tetramer where each of the monomers contributes one helix (helix α2) to a central four-helix bundle (Figure 1). In the tetramer two monomers form an eight-stranded, antiparallel β-sheet with six helices packing against it from one side. The concave, eightstranded β-sheet with its two protruding loops at either end is reminiscent of the saddlelike shape seen in the TATA-box binding protein. The overall dimensions of the tetramer are 60Å x 60Å x 60Å. To probe the relationship between dehydratase activity and transcriptional coactivator functions, the X-ray crystal structures of the free enzyme and its complex with the product analogue 7,8-dihydrobiopterin were solved [105]. The ligand binds at four sites per tetrameric enzyme, with little apparent conformational change in the protein. The pterin binds within an arch of aromatic residues that extends across one dimer interface. The bound ligand makes contacts with the three conserved histidines, and this arrangement restricts proposals for the enzymatic mechanism of dehydratation. PCD/DCoH binds as a dimer to the helical dimerization domain of HNF1-α. A mutant of PCD/DCoH (Cys81→Arg;[115]) with reduced dehydratase activity was not affected in protein-protein interaction and still bound to HNF-1α, showing that enzymatic activity is not essential for HNF1 binding [116]. On the other hand, it was reported that PCD/DCoH retained its enzymatic activity while complexed with HNF1 as a α2β2 heterotetramer [117]. The functional homologue, DCoH2 [102], partially complements PCD/DCoH and, based on crystal structure analysis, adopts as dimer of identical folds with structural differences con- 518 fined largely to the protein surfaces and the tetramer interface [103]. The two paralogues, PDC/DCoH and DCoH2 can even form homotetramers or mixed heterotramers in solution. A functional PCD/DCoH homologue phhB with dehydratase activity as a dimeric enzyme was described from Pseudomonas aeruginosa phhB [118]. The amino acid sequences of the mature human and rat liver proteins are identical, and those of the mouse vary by only one amino acid [99, 101]. Similar proteins for PCD/DCoH and DCoH2 were found in many species throughout the animal kingdom and in various bacteria (e.g. the Cyanobacterium species Synechocystis, Aquifex aeolicus, and Pseudomonas aeruginosa; Figure 3A; see also [103]). DHPR The structure of a binary complex of rat [119] and human DHPR [120] has been determined by X-ray crystallography (Figure 1). DHPR is an α/β protein with a central twisted β-sheet flanked on each side by a layer of α-helices. The β-sheet has seven parallel strands and a single antiparallel strand at one edge leading to the carboxyl terminus of the protein. Connections between individual β-strands involve α-helices. Exceptionally, βB and βC are joined by a short stretch of polypeptide in random-coil conformation. The overall enzyme dimensions are 34Å x 50Å x 73Å. The topology of the backbone folding of DHPR is quite distinct from that of dihydrofolate reductase (DHFR), although the first six strands of the central β-sheet in DHPR have the same overall topological connectivity as that found for the coenzyme-binding domains of several other NAD(P)-dependent dehydrogenases. In contrast to the rat enzyme, human DHPR contains two bound NADH molecules per dimer and despite the sequential amino acid changes, there are only small differences between the two structures (Figure 3B) [120]. Genes encoding the regenerating enzymes PCBD The human PCD/DCoH is encoded by a single copy gene, PCBD, which is located on chromosome 10q22 [121] and composed of four exons [122, 123]. The gene encoding DCoH2 was annotated on chromosome 5, and blast searches identified in addition two pseudogenes on chromosomes 2 and 17 [103]. The exon structures coding for the two human paralogues, PCD/DCoH and DCoH2, are identical and contain 4 exons (for a more detailed comparison of human genes and pseudogenes, see Table 3 in ref. 115). Exon 1 is short in all known PCBD genes from higher eukaryotes, containing only 51, 19, and 23 bp for hen, rat, and human, respectively [124]. Similar as for the human DCoH2 gene, the first exon in PCBD codes for a single amino acid only, the starting methionine, which is separated from the following alanine codon by intronic DNA of more than 2 kb length in all three species. 519 The sequence of the Drosophila melanogaster gene, gpCD1, revealed that it is interrupted by two introns of 82 and 258 nucleotides [125]. Pseudomonas aeruginosa, a gramnegative bacterium, possesses a multi-gene operon that includes a gene encoding a homologue (PhhB) of the regulatory PCD/DCoH, together with genes encoding PAH (PhhA) and aromatic aminotransferase (PhhC) [118, 126]. Within the human PCBD 5’-flanking sequence, potential regulatory regions include consensus-binding sites for transcription factors Sp1, an AP-1, and several AP-2 binding sites; however, the 5’ upstream region lacks both proximal TATA and CAAT box promoter elements [123]. In addition, a comparison of the putative promotor regions between the human, rat, and chicken PCBD genes revealed that all three promoters are located within a region of increased GC content (hen 64%, human 64%, rat 56% [124]). Whether PCBD is transcribed at a basal level by housekeeping factors and further modulated by additional transcriptional elements needs to be determined. QDPR The human QDPR gene is located on chromosome 4p15.3, extends over more than 20 kb, and the coding sequence consists of 732 bp with seven exons ranging within 84–564 bp. Nothing is yet known about the QDPR promoter except that it appears to be GC-rich in sequence [127]. Regulation of enzyme expression and activity PCD/DCoH PCD/DCoH was originally detected as a contaminant in a preparation of rat PAH as a consequence of its ability to stimulate the BH4-dependent hydroxylation of phenylalanine [97]. This stimulating protein was subsequently purified from rat liver [128] and its activity was shown to be due to the catalysis of dehydratation of the 4a-carbinolamine intermediate. The mammalian PCD/DCoH exists in two oligomeric states: as cytoplasmic homotetramer with dehydratase activity and four independent active sites for recycling the BH4-4acarbinolalamine, and as nuclear α2β2 heterodimer with HNF1 proteins [100]. The DCoH2 homologue forms also a homotetramer, displays dehydratase activity, and binds HNF-1 in vivo and in vitro [103]. PCD/DCoH activity was shown to be present in human liver [101], kidney and brain [129], skin [130–132], and hair follicles [133]; its absence is concomitant with the formation of 7-substituted pterins (see Scheme 3). Furthermore, over-expression was demonstrated in human colon cancer and primary melanoma lesions, with unknown etiology in cancerogenesis [134, 135]. On the other hand, extremely low activity of PCD/DCoH was found in patients with the depigmentation disorder vitiligo [132, 136]. PCD/DCoH activity, as a rule, is low in those tissues that contain high levels of tyrosine 520 and tryptophan hydroxylase activity, except for the pineal gland. Analyzing tissues of adult rats with PCD/DCoH-specific antibodies in Western blots, the protein was localized in liver and kidney and in smaller amounts in stomach and intestine [137]. PCD/DCoH is therefore, together with HNF1-α, found in liver and kidney, organs known to express these transcription factors. In liver, all the hepatocytes but not the other cell types are immunoreactive [138]. In kidney, the protein is prevalent in the proximal and distal convoluted tubules, in adrenals, all the cells of the medulla are labeled, and in brain, it generally co-localizes with tyrosine hydroxylase. Positive nerve cells occur in myenteric ganglia of the whole gastrointestinal tract and in the intestinal submucosal ganglia. The prominent nuclear immunoreactivity found in all neural crest cells, but also in other cell types that do not express neither HNF1-α nor aromatic amino acid hydroylases, argue in favor of a new, still unknown function of the protein [138, 139]. Unfortunately, it is not known whether antibodies raised against PCD/DCoH cross-react with its homologue DCoH2. No clear link has so far been established between the enzyme activity of PCD/DCoH and its ability to form stable heterotetramers with HNF1-α. Although PCD/DCoH stabilizes HNF1-α, the enzymatic activity per se is not essential for HNF1-α binding. Using a yeast two-hybrid system it has been shown that naturally occurring substitution mutants of PCD/DCoH with impaired enzymatic activity still bind to HNF1-α ex vivo [116]. Thus, binding to HNF1-α does not interfere with the integrity of the active site of PCD/DCoH. Furthermore, patients with mutant PCD/DCoH with compromised dehydratase activity seem not necessarily to have impaired HNF-1α function [122, 140–142]; however, it is not clear whether the nuclear function, at least for PCD/DCoH null alleles, is complemented by the DCoH2 homologue. In mice lacking HNF1-α the transcriptional rate of genes such as albumin and α1-antitrypsin is reduced, while the gene coding for PAH is totally silent, giving rise to phenylketonuria (PKU) [143]. Mutant mice also suffer from severe Fanconi syndrome caused by renal proximal tubular dysfunction, and are diabetic. In order to prove that PCD/DCoH could enhance the expression of PAH, a number of co-transfection studies were made [144]. PCD/DCoH itself could not transactivate the 9 kb human PAH 5’flanking fragment; however, it was transactivated by HNF1-α in a dose-dependent manner with a maximum of nearly 8-fold activation and PCD/DCoH potentiated this transactivation by another 1.6-fold. These data suggest that the dehydratase can enhance the expression of the human PAH gene. On the other hand, mice lacking PCD/DCoH displayed hyperphenylalaninemia but HNF1-α function was only slightly impaired, leading originally to the identification of the DCoH2 homologue [102,103]. In P. aeruginosa, PhhB, the homologue of PCD/DCoH, is required for in vivo function of phenylalanine hydroxylase (PhhA), and no evidence for a transcriptional role in prokaryotes has been found [126]. The PhhB requirement can be substituted by its mammalian PCD/DCoH counterpart. 521 DHPR The relatively high levels of DHPR, compared with those of aromatic amino acid hydroxylases, and its presence in tissues lacking these enzymes imply that DHPR may be involved in other metabolic processes. For example, there is evidence that DHPR in the presence of NADH could preserve tetrahydrofolate levels in brain where the concentrations of dihydrofolate reductase are low [145]. DHPR is widely distributed in animal tissues [146]. Its occurrence in brain and adrenal medulla is not surprising in view of its role in the tyrosine hydroxylation system in these tissues, and in tryptophan hydroxylation in brain. However, why DHPR should be found in tissues such as heart and lung, which have little or no aromatic amino acid hydroxylating activity, is not clear. DHPR activity has also been detected in cultured fibroblasts, amniocytes, lymphocytes, erythrocytes, and platelets. Detailed studies by immunoprecipitation and two-dimensional electrophoresis showed that DHPR from liver, EBV-transformed lymphoblasts, and fibroblasts are identical [147]. Functions of BH4 Cofactor-dependent enzyme systems The best-investigated function of BH4 is that of its action as a natural cofactor of the aromatic amino acid hydroxylases, phenylalanine-4-hydroxylase (EC 1.14.16.2; PAH), tyrosine-3-hydroxylase (EC 1.14.16.3; TH), and tryptophan-5-hydroxylase (EC 1.14.16.4; TPH), as well as of all three forms of nitric oxide synthase (EC 1.14.13.39; NOS) (for a review see [148]). In addition, BH4 is required by the enzyme glyceryl-ether monooxygenase (EC 1.14.16.5) for hydroxylation of the α-carbon atom of the lipid carbon chain of glyceryl ether to form α-hydroxyalkyl glycerol [149]. The significance of glyceryl-ether monooxygenase in humans has been well documented; however, there is no documentation about the consequences of BH4 deficiency on the alkyl ether metabolism. Enzymatic reactions of aromatic amino acid hydroxylases have been intensively studied and revealed that they have many features in common [150–153]. They all have a strict requirement for dioxygen, iron, and BH4, and the oxidation product of BH4 is released after each catalytic turnover and regenerated by the enzymes PCD/DCoH and DHPR (Scheme 3). The oxidation of BH4 involves the formation of the BH4-4a-carbinolamine intermediate (compound 13), and this has been shown to be formed in reactions of both PAH and TH [98, 154–156]. Studies on PAH found that a stoichiometric amount of BH4 can be oxidized in the presence of oxygen and this yields the reduced enzyme. It has been proposed that this reductive activation of PAH occurs at the redox site and that the enzyme’s iron is a part of this redox site. Its reduction from Fe3+ to Fe2+ has been linked to the formation of active PAH. Two electrons from BH4 are required to reduce the enzyme; one is transferred to Fe3+ and the second apparently to oxygen [157]. The func- 522 tion of BH4 for NOS appears to be different, as the cofactor is a one-electron donor and seems not to dissociate from NOS during turnover (see below). Phenylalanine and BH4 are the major regulators of PAH [157]. While phenylalanine is a positive allosteric effector (activator) that converts inactive enzyme to catalytically competent (activated) enzyme, BH4 is a negative effector that competes with phenylalanine activation to form a dead-end complex PAH.BH4 [158]. Another role of BH4 on the PAH is the chemical chaperon effect preventing protein misfolding and inactivation during dimer/tetramer expression, and protection from proteolytic cleavage [159, 160]. Thus BH4 plays a central regulatory role in the phenylalanine hydroxylating system. The only other known BH4-requiring enzymes in liver, glyceryl-ether monooxygenase and NOS, are present in relatively low amounts, and PAH (subunit) and BH4 concentrations in liver are approximately equal (PAH ~9 µM and BH4 5–10 µM [148, 158–162]). Nevertheless, considering the Km for the cofactor in the PAH (and glyceryl-ether monooxygenase) reaction, which was estimated to be 25–30 µM [161, 163], BH4 is limiting and sub-saturating at least in the liver [160, 164] (with consequences for BH4-responsive PKU; see below). There is no evidence that TH and TPH are regulated by substrate-activated mechanisms similar to those that regulate PAH. All three aromatic amino acid hydroxylases are inhibited by catecholamines, but only the inhibition of human TH is competitive with respect to the BH4 cofactor, and it has been shown that the cofactor can directly displace dopamine from the enzyme active site [165]. BH4 is also utilized as a redox cofactor by all NOS isoforms, inducible NOS (iNOS or NOSII), neuronal NOS (nNOS or NOSI), and endothelial NOS (eNOS or NOSIII). All these NOS share 50–60% sequence homology, and crystal structure determination for several NOS proteins revealed that they have similar homodimeric structures. Each monomer has an N-terminal oxygenase domain with binding sites for arginine, heme and BH4, and a Cterminal reductase domain with binding sites for the cofactors NADPH, FAD, FMN, and Ca2+/calmodulin (for recent reviews see [166, 167]). Two BH4 molecules bind at the dimer interface and are in the vicinity of the arginine substrate binding sites and the heme moieties. NOSs catalyze two sequential monooxygenase reactions, involving NADPH- and O2-dependent hydroxylation of L-arginine via an N-hydroxy-arginine intermediate to NO and L-citrulline. BH4 is required for catalysis but has also structural functions, including dimer stabilization or promoting its formation, protection against proteolysis and increased arginine binding. As mentioned before, for NOSs BH4 is a one-electron donor, and the BH4 radical (BH4+) remains bound in NOS and is subsequently reduced back to BH4 by an electron provided by the NOS reductase domain. In contrast to PAH, requirement for the BH4 cofactor is much lower for the NOS enzyme. The Km values for BH4 for PAH and NOS are 25–30 µM and 0.2–0.3 µM, respectively [168]. Pastor et al. questioned the importance of competition of BH4 between these two hepatic enzymes [169]. They showed that basal BH4 synthesis appears to be adequate to support iNOS activity, whereas BH4 is increased to support PAH activity. Phenylalanine 523 markedly increased BH4 biosynthesis (via GFRP), whereas arginine had no effect. The Km(BH4)-values for the human brain TH range from 0.05–0.1 mM [148], and for the rat brain TPH 30–60 µM [170]. Nevertheless, BH4 (bio-) availability has a critical role when the cofactor is also limiting for NOS activity, as the NOS no longer produces NO but instead becomes uncoupled and generates the superoxide. By uptake of an additional electron, superoxide can form H2O2 or, by reaction with NO, form the cell- or neurotoxic peroxinitrate. Such a mechanism is best documented for the eNOS and endothelial disfunction in insulin-resistant diabetes, but may also play an important role in patients with SR or DHPR deficiencies or induce other pathophysiological pathways [102, 171–175] (for reviews see [63, 92, 176]). Cellular and systemic functions of BH4 In general, BH4 is labile in solution at physiological pH and can readily react with O2 to produce free radicals, thus generating oxidative species in cells, including superoxide, H2O2, and peroxynitrite [177]. On the other hand, BH4 can also protect cells against oxidative damage by scavenging radicals [166]. Spontaneous oxygen reactivity, chaperon-like effect on protein synthesis, superoxide formation by NOS-uncoupling and other functions of BH4 all hint towards the importance of the concentration of BH4 as a critical factor that determines beneficial or adverse effects in vivo. In this context, toxicology of BH4 is also an issue to consider when experimental treatment studies with cells or animals are conducted (for an overview see [178]). One of the earliest discovered cellular functions of BH4 was that as a growth-factor of Crithidia fasciculata. This was initially used to measure biopterins in different body fluids and tissues. More recent observations suggested proliferative activity of BH4 in hemopoietic cells [80, 179]. Exogenous BH4 was found to stimulate DNA synthesis and induce proliferation of some mouse erythroleukemia clonal cell lines. BH4 and sepiapterin also enhanced the proliferation of SV40-transformed human fibroblasts, rat C6 glioma cells, and PC12 cells. Subsequently, it has been shown that epidermal factor (EGF), nerve growth factor (NGF), and insulin-like growth factor-1 (IGF-1) increased the proliferation of rat pheochromocytoma-12 (PC12) cells through obligatory elevation of intracellular BH4 [180, 181]. Besides its proliferative activity, BH4 was also suggested to act as a self-protecting factor for nitric oxide (NO) toxicity with generation of superoxide in NO-producing neurons [182]. Indeed, strong scavenging activity of BH4 against superoxide anion radicals was shown in both xanthine/xanthine oxidase and rat macrophages/phorbol myristate acetate radical-generating systems, and the authors suggested that BH4 might be useful in the treatment of various diseases whose pathogenesis is actively oxygen-related [183]. In another series of experiments, Shimizu et al. [184] demonstrated that S-nitroso-N-acetyl-DL-penicillamine (NO donor)-induced endothelial cell death can be prevented by increasing cellular levels of BH4. This finding was an early observation that suggested the cytotoxicity of NO involving H2O2 production, and that scavenging of H2O2 by BH4 may be at least one of the mechanisms by which BH4 reduces NO-induced endothelial cell death. In contrast, Cho et al. [185] hypothesized that 524 ischemia increases the intracellular BH4 levels and that the increased BH4 level plays a critical role in selective neuronal injury via NOS activation. Using a selective inhibitor of GTPCH in animals exposed to transient forebrain ischemia they demonstrated a marked reduction of BH4 levels, NADPH-diaphorase activity, and caspase-3 gene expression in the CA1 hippocampus. Moreover, delayed neuronal injury in the CA1 hippocampal region was significantly attenuated by the GTPCH inhibitor. These data, in contrast to those presented by Shimizu et al., suggested that a blockade of BH4 biosynthesis may provide novel strategies for neuroprotection. Recently, modulation of BH4-dependent gene expression was put forward based on several observations. In the GTPCH-deficient (hph-1) mouse, up-regulation of liver PAH and brain TH gene expression upon BH4 treatment was reported; however, no such change was found in the wild-type control mice [186]. In smooth-muscle cells, post-transcriptional stabilization of iNOS mRNA was observed indirectly upon varying endogenous BH4 levels [187]. An effect of BH4 supplementation on PAH gene expression was also discussed in BH4-responsive PKU (see below) [188]; however, gene expression or mRNA stability for PAH was not changed, at least not in transgenic mice with different cofactor concentrations present in the liver [160]. Yet another function of BH4 is that it enhances the release of dopamine and serotonin in the rat striatum when administered locally through the dialysis membrane [189, 190]. The enhancement of dopamine release persisted even when dopamine biosynthesis or dopamine re-uptake was completely blocked, but it was abolished when blockers of voltage-dependent Na+ or Ca2+ channels were administered with BH4. Further experiments using selective inhibitors of tyrosine, TH, and NOS demonstrated that BH4 stimulates dopamine release directly, independent of its cofactor action on TH and NOS, by acting from the outside of neurons [191]. The exact mechanism is not entirely clear but it has been shown that arginine also induces a concentration-dependent increase of dopamine release in the superfusate of rat striatum slices, and that it is dependent on the presence of BH4 [192]. BH4 in disease BH4 deficiency is associated with a rare variant of hyperphenylalaninemia that was originally termed “atypical” or “malignant” phenylketonuria (PKU), as it was unresponsive to low-phenylalanine diet (for historical background see other chapters of this book). Today, we differentiate among the BH4 deficiencies between monoamine neurotransmitter deficiency concomitant with hyperphenylalaninemia, i.e. the classical forms of BH4 deficiencies, and monoamine neurotransmitter deficiency without hyperphenylalaninemia, i.e. dopa-responsive dystonia (DRD) and SR deficiency [92]. The classical forms present phenotypically with deficit of the neurotransmitters dopamine and serotonin, and progressive neurological symptoms [193]. It is to be stressed that it is a highly heterogenous group of 525 diseases affecting either all organs, including the central nervous system, or only the peripheral hepatic phenylalanine hydroxylating system [141,142,194]. Classical BH4 deficiency can be caused by mutations in genes encoding enzymes involved in its biosynthesis (GTPCH and PTPS) [86] or regeneration (PCD/DCOH and DHPR) [127, 141, 142]. The mutations are all inherited autosomal-recessively. Biochemical, clinical, and DNA data of patients with BH4 deficiencies are tabulated in the BIODEF and BIOMDB databases and are available on the Internet (www.bh4.org). DRD and SR deficiency are due to a defective BH4 biosynthesis that occurs without hyperphenylalaninemia. DRD, initially described as Segawa disease, may be caused by autosomal dominant mutations in the GCH gene, but about 40% of the patients present without identifiable DNA mutations. Furthermore, most of these inherited dominant alleles have no penetrance in the parent of the patient [195–199]. In contrast, SR deficiency is inherited as an autosomal recessive trait, and exhibits biochemical abnormalities that are related to disease exclusively in neurotransmitter metabolites and pterins in the cerebrospinal fluid (CSF) [17, 92]. As mentioned before, SR deficiency suggested the presence of an alternative biosynthetic pathway operative in peripheral tissues and involving alternative aldose-ketose reductases and the dihydrofolate reductase. Decreased levels of BH4 in the CSF have also been documented in other neurological diseases presenting phenotypically without hyperphenylalaninemia, such as Parkinson’s disease [200], autism [201], depression [202], and Alzheimer’s disease [203]. In some of these, administration of BH4 has been reported to improve the clinical symptoms [200, 204, 205]. Unfortunately, others were not able to confirm these results [189], and thus a benefit of BH4 therapy at least for this group of diseases is questionable. Another group of diseases with perturbed BH4 metabolism in human epidermis are skin disorders, including vitiligo and Hermansky-Pudlak syndrome. Although the etiology of these disorders is not yet known, both involve lowered PCD/DCoH activities concomitant with 6- and 7-biopterin and H2O2 accumulation in skin, tyrosinase inhibition, and abnormal melanin biosynthesis [132, 206–208]. Finally, a group of mild phenylketonuric patients with autosomal recessive mutations in the gene encoding PAH but with normal BH4 metabolism respond to treatment with oral BH4 as an alternative to a low-phenylalanine diet [178, 209]. Recent investigations on the mechanism of this BH4-responsive PKU revealed a multi-factorial basis, including a chemical chaperon effect of BH4 that prevents degradation of mutant PAH-proteins and protection from rapid inactivation. Furthermore, some mutants show a kinetic defect with increased Km for BH4 [159, 210]. In any case, a PAH allele with residual activity is a prerequisite for response. On the other hand, PAH gene expression or mRNA stability seems not to be affected by hepatic BH4 cofactor concentration [160]. For more details on diseases due to BH4 deficiencies see other chapters in this book. 526 Animal models to study BH4 function and deficiency Today, three different animal models are available for BH4 deficiencies; these are the hph-1 mouse and two different knock-out strains for PTPS. The hph-1 mouse was produced by screening ethylnitrosourea (ENU)-treated mice for the presence of hyperphenylalaninemia [211]. This chemically induced mutant strain has a mild, neonatal and transient form of hyperphenylalaninemia with lowered GTPCH activity and BH4 cofactor levels in the liver. In the brain, the mouse showed low levels of BH4, catecholamines, serotonin, and TH activity. Thus, biochemical analysis revealed that this strain provides an animal model for DRD [212]. Unfortunately no DNA mutation could be identified thus far, but genomic mapping narrowed the site of mutation to an interval containing the mouse GCH1 gene [213]. Two PTPS-deficient strains were generated by targeted Pts-gene disruption. The mouse presented with perinatal lethality due to monoamine neurotransmitter depletion, but rescue experiments with neurotransmitter precursors and/or BH4 revealed that liver PAH, and brain TH and TPH might be regulated differentially [160, 214, 215]. This Pts-knock-out mouse is thus a valuable tool to investigate BH4-deficiency and their consequences for all BH4-dependent enzyme systems. Acknowledgements I would like to thank Günter Auerbach for help with updates on structural aspects, David Meili and Andreas Schweizer for preparing the figures, Nenad Blau for valuable discussions, and Margrith Killen for helping with the preparation of the manuscript. REFERENCES: 1. Thöny B, Auerbach G, and Blau N. (2000) Tetrahydrobiopterin biosynthesis, regeneration and functions Biochem. J. 347 Pt 1, 1–16. 2. Werner-Felmayer G, Golderer G, and Werner ER. (2002) Tetrahydrobiopterin biosynthesis, utilization and pharmacological effects Curr Drug Metab 3, 159–173. 3. Bracher A, Eisenreich W, Schramek N, Ritz H, Gotze E, Herrmann A, Gutlich M and Bacher A. (1998) Biosynthesis of pteridines. NMR studies on the reaction mechanisms of GTP cyclohydrolase I, pyruvoyltetrahydropterin synthase, and sepiapterin reductase J Biol Chem 273, 28132–28141. 4. Nar H, Huber R, Meining W, Schmid C, Winkauf S, and Bacher A. (1995) Atomic structure of the GTP cyclohydrolase I Structure 3, 459–466. 527 5. Auerbach G, Herrmann A, Bracher A, Bader G, Gutlich M, Fischer M, Neukamm M, Garrido-Franco M, Richardson J, Nar H, Huber R, and Bacher A. (2000) Zinc plays a key role in human and bacterial GTP cyclohydrolase I Proc Natl Acad Sci U S A 97, 13567–13572. 6. Rebelo J, Auerbach G, Bader G, Bracher A, Nar H, Hosl C, Schramek N, Kaiser J, Bacher A, Huber R, and Fischer M. (2003) Biosynthesis of pteridines. Reaction mechanism of GTP cyclohydrolase I J Mol Biol 326, 503–516. 7. Schramek N, Bracher A, Fischer M, Auerbach G, Nar H, Huber R, and Bacher A. (2002) Reaction mechanism of GTP cyclohydrolase I: single turnover experiments using a kinetically competent reaction intermediate J Mol Biol 316, 829–837. 8. Nar H, Huber R, Heizmann CW, Thöny B, and Bürgisser D. (1994) Three-dimensional structure of 6-pyruvoyl tetrahydropterin synthase, an enzyme involved in tetrahydrobiopterin biosynthesis EMBO Journal 13, 1255–1262. 9. Bürgisser D, Thöny B, Redweik U, Hess D, Heizmann CW, Huber R, and Nar H. (1995) 6-Pyruvoyl tetrahydropterin synthase: an enzyme with a novel type of active site involving both zinc binding and an intersubunit catalytic motif; site-directed mutagenesis of the proposed active center, characterization of the metal binding site and modelling of substrate binding J Mol Biol 253, 358–369. 10. Ploom T, Thöny B, Lee S, Nar H, Leimbacher W, Huber R, and Auerbach G. (1999) Crystallographic and kinetic investigations on the mechanism of 6-pyruvoyl-tetrahydropterin synthase J Mol Biol 286, 851–860. 11. Auerbach G, Herrmann A, Gütlich M, Fischer M, Jacob U, Bacher A, and Huber R. (1997) The 1.25 A crystal structure of sepiapterin reductase reveals its binding mode to pterins and brain neurotransmitters EMBO Journal 16, 7219–7230. 12. Fujimoto K, Takahashi SY, and Katoh S. (2002) Mutational analysis of sites in sepiapterin reductase phosphorylated by Ca2+/calmodulin-dependent protein kinase II Biochim Biophys Acta 1594, 191–198. 13. Nichol CA, Smith GK, and Duch DS. (1985) Biosynthesis and metabolism of tetrahydrobiopterin and molybdopterin Ann Rev Biochem 54, 729–764. 14. Milstien S, and Kaufman S. (1989) The biosynthesis of tetrahydrobiopterin in rat brain. Purification and characterization of 6-pyruvoyl tetrahydropterin (2’-oxo)reductase J Biol Chem 264, 8066–80673. 15. Park YS, Heizmann CW, Wermuth B, Levine RA, Steinerstauch P, Guzman J, and Blau N. (1991) Human carbonyl and aldose reductases: new catalytic functions in tetrahydrobiopterin biosynthesis Biochem Biophys Res Commun 175, 738–744. 528 16. Iino T, Tabata M, Takikawa S, Sawada H, Shintaku H, Ishikura S, and Hara A. (2003) Tetrahydrobiopterin is synthesized from 6-pyruvoyl-tetrahydropterin by the human aldo-keto reductase AKR1 family members Arch Biochem Biophys 416, 180–187. 17. Bonafé L, Thöny B, Penzien JM, Czarnecki B, and Blau N. (2001) Mutations in the sepiapterin reductase gene cause a novel tetrahydrobiopterin-dependent monoamine-neurotransmitter deficiency without hyperphenylalaninemia Am J Hum Genet 69, 269–277. 18. Nar H, Huber R, Auerbach G, Fischer M, Hösl C, Ritz H, Bracher A, Meining W, Eberhardt S, and Bacher A. (1995) Active site topology and reaction mechanism of GTP cyclohydrolase I Proc Natl Acad Sci USA 92, 12120–12125. 19. Maita N, Okada K, Hatakeyama K, and Hakoshima T. (2002) Crystal structure of the stimulatory complex of GTP cyclohydrolase I and its feedback regulatory protein GFRP Proc Natl Acad Sci USA 99, 1212–1217. 20. Bader G, Schiffmann S, Herrmann A, Fischer M, Gutlich M, Auerbach G, Ploom T, Bacher A, Huber R, and Lemm T. (2001) Crystal structure of rat GTP cyclohydrolase I feedback regulatory protein, GFRP J Mol Biol 312, 1051–1057. 21. Auerbach G, and Nar H. (1997) The pathway from GTP to tetrahydropbiopterin: three-dimensional structures of GTP cyclohydrolase I and 6-pyruvoyl-tetrahydropterin synthase Biol Chem 378, 185–192. 22. Ploom T, Haussmann C, Hof P, Steinbacher S, Bacher A, Richardson J, and Huber R. (1999) Crystal structure of 7,8-dihydroneopterin triphosphate epimerase Structure Fold Des 7, 509–516. 23. Ichinose H, Ohye T, Matsuda Y, Hori T, Blau N, Burlina A, Rouse B, Matalon R, Fujita K, and Nagatsu T. (1995) Characterization of mouse and human GTP cyclohydrolase I genes. Mutations in patients with GTP cyclohydrolase I deficiency J Biol Chem 270, 10062–10071. 24. McLean JR, Krishnakumar S, and O’Donnell JM. (1993) Multiple mRNAs from the Punch locus of Drosophila melanogaster encode isoforms of GTP cyclohydrolase I with distinct N-terminal domains J Biol Chem 268, 27191–27197. 25. Witter K, Cahill DJ, Werner T, Ziegler I, Rodl W, Bacher A, and Gutlich M. (1996) Molecular cloning of a cDNA coding for GTP cyclohydrolase I from Dictyostelium discoideum Biochem J 319 ( Pt 1), 27–32. 26. Nardese V, Gutlich M, Brambilla A, and Carbone ML. (1996) Disruption of the GTPcyclohydrolase I gene in Saccharomyces cerevisiae Biochem Biophys Res Commun 218, 273–279. 529 27. Maier J, Witter K, Gutlich M, Ziegler I, Werner T, and Ninnemann H. (1995) Homology cloning of GTP-cyclohydrolase I from various unrelated eukaryotes by reversetranscription polymerase chain reaction using a general set of degenerate primers Biochem Biophys Res Commun 212, 705–711. 28. Togari A, Ichinose H, Matsumoto S, Fujita K, and Nagatsu T. (1992) Multiple mRNA forms of human GTP cyclohydrolase I Biochem Biophys Res Commun 187, 359–365. 29. Golderer G, Werner ER, Heufler C, Strohmaier W, Grobner P, and Werner-Felmayer G. (2001) GTP cyclohydrolase I mRNA: novel splice variants in the slime mould Physarum polycephalum and in human monocytes (THP-1) indicate conservation of mRNA processing Biochem J 355, 499–507. 30. Hatakeyama K, Inoue Y, Harada T, and Kagamiyama H. (1991) Cloning and sequencing of cDNA encoding rat GTP cyclohydrolase I J Biol Chem 266, 765–769. 31. Nomura T, Ichinose H, Sumi-Ichinose C, Nomura H, Hagino Y, Fujita K, and Nagatsu T. (1993) Cloning and sequencing of cDNA encoding mouse GTP cyclohydrolase I Biochem Biophys Res Commun 191, 523–527. 32. Nomura T, Ohtsuki M, Matsui S, Sumi-Ichinose C, Nomura H, Hagino Y, Iwase K, Ichinose H, Fujita K, and Nagatsu T. (1995) Isolation of a full-length cDNA clone for human GTP cyclohydrolase I type 1 from pheochromocytoma J Neural Transm-Gen Sect 101, 237–242. 33. Hwu WL, Yeh HY, Fang SW, Chiang HS, Chiou YW, and Lee YM. (2003) Regulation of GTP cyclohydrolase I by alternative splicing in mononuclear cells Biochem Biophys Res Commun 306, 937–942. 34. Hwu WL, Chiou YW, Lai SY, and Lee YM. (2000) Dopa-responsive dystonia is induced by a dominant-negative mechanism Ann Neurol 48, 609–613. 35. Sakamoto H, Fujita K, Ikegame M, Teradaira R, and Kuzuya H. (1996) Different complex forms of human liver GTP cyclohydrolase I Biogenic Amines 12, 55–67. 36. Ichinose H, Ohye T, Matsuda Y, Hori T, Blau N, Burlina A, Rouse B, Matalon R, Fujita K, and Nagatsu T. (1995) Characterization of mouse and human GTP cyclohydrolase I genes – mutations in patients with GTP cyclohydrolase I deficiency J Biol Chem 270, 10062–10071. 37. Witter K, Werner T, Blusch JH, Schneider EM, Riess O, Ziegler I, Rodl W, Bacher A, and Gutlich M. (1996) Cloning, sequencing and functional studies of the gene encoding human GTP cyclohydrolase I Gene 171, 285–290. 38. Kapatos G, Stegenga SL, and Hirayama K. (2000) Identification and characterization of basal and cyclic AMP response elements in the promoter of the rat GTP cyclohydrolase I gene J Biol Chem 275, 5947–5957. 530 39. Hirayama K, Shimoji M, Swick L, Meyer A, and Kapatos G. (2001) Characterization of GTP cyclohydrolase I gene expression in the human neuroblastoma SKNBE(2)M17: enhanced transcription in response to cAMP is conferred by the proximal promoter J Neurochem 79, 576–587. 40. Suzuki T, Inagaki H, Yamakuni T, Nagatsu T, and Ichinose H. (2002) Enhanced expression of GTP cyclohydrolase I in V-1-overexpressing PC12D cells Biochem Biophys Res Commun 293, 962–968. 41. Wu SM, Kuo WC, Hwu WL, Hwa KY, Mantovani R, and Lee YM. (2004) RNF4 is a coactivator for nuclear factor Y on GTP cyclohydrolase I proximal promoter Mol Pharmacol 66, 1317–1324. 42. Kluge C, Brecevic L, Heizmann CW, Blau N, and Thöny B. (1996) Chromosomal localization, genomic structure and characterization of the human gene and a retropseudogene for 6-pyruvoyltetrahydropterin synthase Eur J Biochem 240, 477–484. 43. Kluge K, Leimbacher W, Heizmann CW, Blau N, and Thöny B. (1996) Isolation of 6pyruvoyl-tetrahydropterin synthase cDNAs from human brain Pteridines 7, 91–93. 44. Turri MO, Ilg EC, Thöny B, and Blau N. (1998) Structure, genomic localization and recombinant expression of the mouse 6-pyruvoyl-tetrahydropterin synthase gene Biol Chem 379, 1441–1447. 45. Leitner KL, Meyer M, Leimbacher W, Peterbauer A, Hofer S, Heufler C, Muller A, Heller R, Werner ER, Thöny B, and Werner-Felmayer G. (2003) The low tetrahydrobiopterin biosynthetic capacity of human monocytes is caused by exon skipping in 6-pyruvoyl tetrahydropterin synthase Biochem J 373, 681–688. 46. Kim N, Kim J, Park D, Rosen C, Dorsett D, and Yim J. (1996) Structure and expression of wild-type and suppressible alleles of the Drosophila purple gene Genetics 142, 1157–1168. 47. Ohye T, Hori TA, Katoh S, Nagatsu T, and Ichinose H. (1998) Genomic organization and chromosomal localization of the human sepiapterin reductase gene Biochem Biophys Res Commun 251, 597–602. 48. Lee SW, Park IY, Hahn Y, Lee JE, Seong CS, Chung JH, and Park YS. (1999) Cloning of mouse sepiapterin reductase gene and characterization of its promoter region Biochim Biophys Acta 1445, 165–171. 49. Seong C, Baek K, and Yoon J. (2000) Structure, chromosomal localization, and expression of the Drosophila melanogaster gene encoding sepiapterin reductase Gene 255, 357–361. 50. Milstien S, Jaffe H, Kowlessur D, and Bonner TI. (1996) Purification and cloning of the GTP cyclohydrolase I feedback regulatory protein, GFRP J Biol Chem 271, 19743–19751. 531 51. Werner ER, Bahrami S, Heller R, and Werner-Felmayer G. (2002) Bacterial lipopolysaccharide down-regulates expression of GTP cyclohydrolase I feedback regulatory protein J Biol Chem 277, 10129–10133. 52. Gesierich A, Niroomand F, and Tiefenbacher CP. (2003) Role of human GTP cyclohydrolase I and its regulatory protein in tetrahydrobiopterin metabolism Basic Res Cardiol 98, 69–75. 53. Larsen F, Gundersen G, Lopez R, and Prydz H. (1992) CpG islands as gene markers in the human genome Genomics 13, 1095–1107. 54. Gardiner-Garden M, and Frommer M. (1987) CpG islands in vertebrate genomes J Mol Biol 196, 261–282. 55. Dassesse D, Hemmens B, Cuvelier L, and Resibois A. (1997) GTP cyclohydrolase Ilike immunoreactivity in rat brain Brain Res 777, 187–201. 56. Vann LR, Payne SG, Edsall LC, Twitty S, Spiegel S, and Milstien S. (2002) Involvement of sphingosine kinase in TNF-alpha-stimulated tetrahydrobiopterin biosynthesis in C6 glioma cells J Biol Chem 277, 12649–12656. 57. Skimming JW, Nasiroglu O, Huang CJ, Wood CE, Stevens BR, Haque IU, Scumpia PO, and Sarcia PJ. (2003) Dexamethasone suppresses iNOS yet induces GTPCH and CAT-2 mRNA expression in rat lungs Am J Physiol Lung Cell Mol Physiol 285, L484–491. 58. Kaneko YS, Mori K, Nakashima A, Nagatsu I, and Ota A. (2003) Peripheral administration of lipopolysaccharide enhances the expression of guanosine triphosphate cyclohydrolase I mRNA in murine locus coeruleus Neurosci 116, 7–12. 59. Bauer M, Suppmann S, Meyer M, Hesslinger C, Gasser T, Widmer HR, and Ueffing M. (2002) Glial cell line-derived neurotrophic factor up-regulates GTP-cyclohydrolase I activity and tetrahydrobiopterin levels in primary dopaminergic neurones J Neurochem 82, 1300–1310. 60. Zhu M, Hirayama K, and Kapatos G. (1994) Regulation of tetrahydrobiopterin biosynthesis in cultured dopamine neurons by depolarization and cAMP J Biol Chem 269, 11825–11829. 61. Hirayama K, Lentz SI, and Kapatos G. (1993) Tetrahydrobiopterin cofactor biosynthesis: GTP cyclohydrolase I mRNA expression in rat brain and superior cervical ganglia J Neurochem 61, 1006–1014. 62. Serova LI, Maharjan S, Huang A, Sun D, Kaley G, and Sabban EL. (2004) Response of tyrosine hydroxylase and GTP cyclohydrolase I gene expression to estrogen in brain catecholaminergic regions varies with mode of administration Brain Res 1015, 1–8. 532 63. Shi W, Meininger CJ, Haynes TE, Hatakeyama K, and Wu G. (2004) Regulation of tetrahydrobiopterin synthesis and bioavailability in endothelial cells Cell Biochem Biophys 41, 415–434. 64. Shimizu S, Shiota K, Yamamoto S, Miyasaka Y, Ishii M, Watabe T, Nishida M, Mori Y, Yamamoto T, and Kiuchi Y. (2003) Hydrogen peroxide stimulates tetrahydrobiopterin synthesis through the induction of GTP-cyclohydrolase I and increases nitric oxide synthase activity in vascular endothelial cells Free Radic Biol Med 34, 1343–1352. 65. Serova L, Nankova B, Rivkin M, Kvetnansky R, and Sabban EL. (1997) Glucocorticoids elevate GTP cyclohydrolase I mRNA levels in vivo and in PC12 cells Brain Res Mol Brain Res 48, 251–258. 66. Simmons WW, Ungureanu-Longrois D, Smith GK, Smith TW, and Kelly RA. (1996) Glucocorticoids regulate inducible nitric oxide synthase by inhibiting tetrahydrobiopterin synthesis and L-arginine transport J Biol Chem 271, 23928–23937. 67. Werner ER, Werner-Felmayer G, and Mayer B. (1998) Tetrahydrobiopterin, cytokines, and nitric oxide synthesis Proc Soc Exp Biol Med 219, 171–182. 68. Lapize C, Pluss C, Werner ER, Huwiler A, and Pfeilschifter J. (1998) Protein kinase C phosphorylates and activates GTP cyclohydrolase I in rat renal mesangial cells Biochem Biophys Res Commun 251, 802–805. 69. Imazumi K, Sasaki T, Takahashi K, and Takai Y. (1994) Identification of a rabphilin3A-interacting protein as GTP cyclohydrolase I in PC12 cells Biochem Biophys Res Commun 205, 1409–1416. 70. Hesslinger C, Kremmer E, Hultner L, Ueffing M, and Ziegler I. (1998) Phosphorylation of GTP cyclohydrolase I and modulation of its activity in rodent mast cells. GTP cyclohydrolase I hyperphosphorylation is coupled to high affinity IgE receptor signaling and involves protein kinase C J Biol Chem 273, 21616–21622. 71. Harada T, Kagamiyama H, and Hatakeyama K. (1993) Feedback regulation mechanism for the control of GTP cyclohydrolase I activity Science 260, 1507–1510. 72. Yoneyama T, Brewer JM, and Hatakeyama K. (1997) GTP cyclohydrolase I feedback regulatory protein is a pentamer of identical subunits J Biol Chem 272, 9690–9696. 73. Yoneyama T, and Hatakeyama K. (1998) Decameric GTP cyclohydrolase I forms complexes with two pentameric GTP cyclohydrolase I feedback regulatory proteins in the presence of phenylalanine or of a combination of tetrahydrobiopterin and GTP J Biol Chem 273, 20102–20108. 74. Yoneyama T, and Hatakeyama K. (2001) Ligand binding to the inhibitory and stimulatory GTP cyclohydrolase I/GTP cyclohydrolase I feedback regulatory protein complexes Protein Sci 10, 871–878. 533 75. Steinmetz MO, Pluss C, Christen U, Wolpensinger B, Lustig A, Werner ER, Wachter H, Engel A, Aebi U, Pfeilschifter J, and Kammerer RA. (1998) Rat GTP cyclohydrolase I is a homodecameric protein complex containing high-affinity calcium-binding sites J Mol Biol 279, 189–199. 76. Kapatos G, Hirayama K, Shimoji M, and Milstien S. (1999) GTP cyclohydrolase I feedback regulatory protein is expressed in serotonin neurons and regulates tetrahydrobiopterin biosynthesis J Neurochem 72, 6696–75. 77. Kolinsky MA, and Gross SS. (2004) The mechanism of potent GTP cyclohydrolase I inhibition by 2,4-diamino-6-hydroxypyrimidine: requirement of the GTP cyclohydrolase I feedback regulatory protein J Biol Chem 279, 40677–40682. 78. Werner ER, Werner-Felmayer G, Fuchs D, Hausen A, Reibnegger G, Yim JJ, Pfleiderer W, and Wachter H. (1990) Tetrahydrobiopterin biosynthetic activities in human macrophages, fibroblasts, THP-1, and T 24 cells. GTP-cyclohydrolase I is stimulated by interferon-gamma, and 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase are constitutively present J Biol Chem 265, 3189–3192. 79. Werner ER, Werner-Felmayer G, Fuchs D, Hausen A, Reibnegger G, Yim JJ, and Wachter H. (1991) Impact of tumour necrosis factor-alpha and interferon-gamma on tetrahydrobiopterin synthesis in murine fibroblasts and macrophages Biochem J 280 ( Pt 3), 709–714. 80. Kerler F, Ziegler I, Schwarzkopf B, and Bacher A. (1989) Regulation of tetrahydrobiopterin synthesis during lectin stimulation of human peripheral blood lymphocytes FEBS Lett 250, 622–624. 81. Ziegler I, Schott K, Lubbert M, Herrmann F, Schwulera U, and Bacher A. (1990) Control of tetrahydrobiopterin synthesis in T lymphocytes by synergistic action of interferon-gamma and interleukin-2 J Biol Chem 265, 17026–17030. 82. Werner-Felmayer G, Prast H, Werner ER, Philippu A, and Wachter H. (1993) Induction of GTP cyclohydrolase I by bacterial lipopolysaccharide in the rat FEBS Lett 322, 223–226. 83. Linscheid P, Schaffner A, Blau N, and Schoedon G. (1998) Regulation of 6-pyruvoyltetrahydropterin synthase activity and messenger RNA abundance in human vascular endothelial cells Circulation 98, 1703–1706. 84. Franscini N, Blau N, Walter RB, Schaffner A, and Schoedon G. (2003) Critical Role of interleukin-1{beta} for transcriptional regulation of endothelial 6-pyruvoyltetrahydropterin synthase Arterioscler Thromb Vasc Biol 23, E50–E53. 85. Hirayama K, and Kapatos G. (1995) Expression and regulation of rat 6-pyruvoyl tetrahydropterin synthase mRNA Neurochem Int 26, 601–606. 534 86. Thöny B, and Blau N. (1997) Mutations in the GTP cyclohydrolase I and 6-pyruvoyltetrahydropterin synthase genes Hum Mutat 10, 11–20. 87. Inoue Y, Kawasaki Y, Harada T, Hatakeyama K, and Kagamiyama H. (1991) Purification and cDNA cloning of rat 6-pyruvoyl-tetrahydropterin synthase J Biol Chem 266, 20791–20796. 88. Scherer-Oppliger T, Leimbacher W, Blau N, and Thöny B. (1999) Serine 19 of human 6-pyruvoyl-tetrahydropterin synthase is a substrate for cGMP-protein kinase IIdependent phosphorylation J Biol Chem 274, 31341–31348. 89. Oppliger T, Thöny B, Nar H, Burgisser D, Huber R, Heizmann CW, and Blau N. (1995) Structural and functional consequences of mutations in 6-pyruvoyltetrahydropterin synthase causing hyperphenylalaninemia in humans. Phosphorylation is a requirement for in vivo activity J Biol Chem 270, 29498–29506. 90. Ikemoto K, Suzuki T, Ichinose H, Ohye T, Nishimura A, Nishi K, Nagatsu I, and Nagatsu T. (2002) Localization of sepiapterin reductase in the human brain Brain Res 954, 237–246. 91. Fujimoto K, Sakurai M, Kawase M, and Katoh S. (2003) Effect of antisense oligodeoxynucleotide for sepiapterin reductase on the viability of PC12 cells in the presence of exogenous carbonyl compounds Chem Biol Interact 143–144, 583–586. 92. Blau N, Bonafe L, and Thöny B. (2001) Tetrahydrobiopterin deficiencies without hyperphenylalaninemia: diagnosis and genetics of dopa-responsive dystonia and sepiapterin reductase deficiency Mol Genet Metab 74, 172–185. 93. Oyama R, Katoh S, Sueoka T, Suzuki M, Ichinose H, Nagatsu T, and Titani K. (1990) The complete amino acid sequence of the mature form of rat sepiapterin reductase Biochem Biophys Res Commun 173, 627–631. 94. Katoh S, Sueoka T, Yamamoto Y, and Takahashi SY. (1994) Phosphorylation by Ca(II)/calmodulin-dependent protein kinase II and protein kinase C of sepiapterin reductase, the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin FEBS Lett 341, 227–232. 95. Itagaki C, Isobe T, Taoka M, Natsume T, Nomura N, Horigome T, Omata S, Ichinose H, Nagatsu T, Greene LA, and Ichimura T. (1999) Stimulus-coupled interaction of tyrosine hydroxylase with 14-3-3 proteins Biochem 38, 15673–15680. 96. Elzaouk L, Laufs S, Heerklotz D, Leimbacher W, Blau N, Resibois A, and Thony B. (2004) Nuclear localization of tetrahydrobiopterin biosynthetic enzymes Biochim Biophys Acta 1670, 56–68. 97. Kaufman S. (1970) A protein that stimulates rat liver phenylalanine hydroxylase J Biol Chem 254, 4751–4759. 535 98. Lazarus RA, Benkovic SJ, and Kaufman S. (1983) Phenylalanine hydroxylase stimulator protein is a 4a-carbinolamine dehydratase J Biol Chem 258, 10960–10962. 99. Mendel D, Kahavari PA, Conley PB, Graves MK, Hansen LP, Admon A, and Crabtree GR. (1991) Characterization of a cofactor that regulates dimerization of a mammalian homeodomain protein Science 254, 1762–1767. 100. Citron BA, Davis MD, Milstien S, Gutierrez J, Mendel DB, Crabtree GR, and Kaufman S. (1992) Identity of 4a-carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, and DCoH, a transregulator of homeodomain proteins Proc Natl Acad Sci USA 89, 11891–11894. 101. Hauer CR, Rebrin I, Thony B, Neuheiser F, Curtius HC, Hunziker P, Blau N, Ghisla S, and Heizmann CW. (1993) Phenylalanine hydroxylase-stimulating protein/pterin-4 alpha-carbinolamine dehydratase from rat and human liver. Purification, characterization, and complete amino acid sequence J Biol Chem 268, 4828–4831. 102. Bayle JH, Randazzo F, Johnen G, Kaufman S, Nagy A, Rossant J, and Crabtree GR. (2002) Hyperphenylalaninemia and impaired glucose tolerance in mice lacking the bifunctional DCoH gene J Biol Chem 277, 28884–28891. 103. Rose RB, Pullen KE, Bayle JH, Crabtree GR, and Alber T. (2004) Biochemical and structural basis for partially redundant enzymatic and transcriptional functions of DCoH and DCoH2 Biochem 43, 7345–7355. 104. Rebrin I, Bailey SW, Boerth SR, Ardell MD, and Ayling JE. (1995) Catalytic characterization of 4a-hydroxytetrahydropterin dehydratase Biochem 34, 5801–5810. 105. Cronk JD, Endrizzi JA, and Alber T. (1996) High-resolution structures of the bifunctional enzyme and transcriptional coactivator DCoH and its complex with a product analogue Prot Sci 5, 1963–1972. 106. Köster S, Thöny B, Macheroux P, Curtius, HC, Heizmann CW, Pfleiderer W, and Ghisla S. (1995) Human pterin-4a-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor-1a: characterization and kinetic analysis of wild-type and mutant enzymes Eur J Biochem 231, 414–423. 107. Rebrin I, Thony B, Bailey SW, and Ayling JE. (1998) Stereospecificity and catalytic function of histidine residues in 4a-hydroxy-tetrahydropterin dehydratase/DCoH Biochem 37, 11246–11254. 108. Köster S, Stier G, Ficner R, Holzer M, Curtius HC, Suck D, and Ghisla S. (1996) Location of the active site and proposed catalytic mechanism of pterin-4a-carbinolamine dehydratase Eur J Biochem 241, 858–864. 536 109. Bailey SW, Boerth SR, Dillard SB, and Ayling JE. (1993) The mechanism of cofactor regeneration during phenylalanine hydroxylation Advances in Experimental Medicine and Biology: Chemistry and Biology of Pteridines and Folates. 338, 47–54, Plenum Press. 110. Curtius HC, Adler C, Rebrin I, Heizmann CW, and Ghisla S. (1990) 7-Substituted pterins: formation during phenylalanine hydroxylation in the absence of dehydratase Biochem Biophys Res Commun 172, 1060–1066. 111. Davis MD, Kaufman S, and Milstien S. (1991) Conversion of 6-substituted tetrahydropterins to 7-isomers via phenylalanine hydroxylase-generated intermediates Proc Natl Acad Sci USA 88, 385–389. 112. Armarego WL, Randles D, and Waring P. (1984) Dihydropteridine reductase (DHPR), its cofactors, and its mode of action Med Res Rev 4, 267–321. 113. Ficner R, Sauer UH, Stier G, and Suck D. (1995) Three-dimensional structure of the bifunctional protein PCD/DCoH, a cytoplasmic enzyme interacting with transcription factor HNF1 Embo J 14, 2034–2042. 114. Endrizzi JA, Cronk JD, Wang W, Crabtree GR, and Alber T. (1995) Crystal structure of DCoH, a bifunctional, protein-binding transcriptional coactivator Science 268, 556–559. 115. Köster S, Thöny B, Macheroux P, Curtius HC, Heizmann CW, Pfleiderer W, and Ghisla S. (1995) Human pterin-4 alpha-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor-1 alpha. Characterization and kinetic analysis of wildtype and mutant enzymes Eur J Biochem 231, 414–423. 116. Sourdive DJ, Transy C, Garbay S, and Yaniv M. (1997) The bifunctional DCOH protein binds to HNF1 independently of its 4-alpha-carbinolamine dehydratase activity Nucleic Acids Res 25, 1476–1484. 117. Rhee KH, Stier G, Becker PB, Suck D, and Sandaltzopoulos R. (1997) The bifunctional protein DCoH modulates interactions of the homeodomain transcription factor HNF1 with nucleic acids J Mol Biol 265, 20–29. 118. Zhao G, Xia T, Song J, and Jensen RA. (1994) Pseudomonas aeruginosa possesses homologues of mammalian phenylalanine hydroxylase and 4 alpha-carbinolamine dehydratase/DCoH as part of a three-component gene cluster Proc Natl Acad Sci USA 91, 1366–1370. 119. Varughese KI, Skinner MM, Whiteley JM, Matthews DA, and Xuong NH. (1992) Crystal structure of rat liver dihydropteridine reductase Proc Natl Acad Sci USA 89, 6080–6084. 537 120. Su Y, Varughese KI, Xuong NH, Bray TL, Roche DJ, and Whiteley JM. (1993) The crystallographic structure of a human dihydropteridine reductase NADH binary complex expressed in Escherichia coli by a cDNA constructed from its rat homologue J Biol Chem 268, 26836–26841. 121. Thöny B, Heizmann CW, and Mattei MG. (1994) Chromosomal location of two human genes encoding tetrahydrobiopterin-metabolizing enzymes: 6-pyruvoyltetrahydropterin synthase maps to 11q22.3-q23.3, and pterin-4 alpha-carbinolamine dehydratase maps to 10q22 Genomics 19, 365–368. 122. Citron BA, Kaufman S, Milstien S, Naylor EW, Greene CL, and Davis MD. (1993) Mutation on the 4a-carbinolamine dehydratase gene leads to mild hyperphenylalaninemia with defective cofactor metabolism Am J Hum Gen 53, 768–774. 123. Thöny B, Neuheiser F, Blau N, and Heizmann CW. (1995) Characterization of the human PCBD gene encoding the bifunctional protein pterin-4a-carbinolamine dehydratase/dimerization cofactor for the transcription factor HNF-1a Biochem Biophys Res Commun 210, 966–973. 124. Bossow S, Riepl S, and Igo-Kemenes T. (1998) Characterization of the chicken and rat DCoH gene domains using an improved ligation-mediated PCR method Biol Chem 379, 1359–1365. 125. Seong C, Jeong S, Park D, Yoon J, Oh Y, Yim J, Han K, and Baek K. (1998) Molecular characterization of the Drosophila melanogaster gene encoding the pterin 4alpha-carbinolamine dehydratase Biochim Biophys Acta 1388, 273–278. 126. Song J, Xia T, and Jensen RA. (1999) PhhB, a Pseudomonas aeruginosa homolog of mammalian pterin 4a-carbinolamine dehydratase/DCoH, does not regulate expression of phenylalanine hydroxylase at the transcriptional level J Bacteriol 181, 2789–2796. 127. Dianzani I, de Sanctis L, Smooker PM, Gough TJ, Alliaudi C, Brusco A, Spada M, Blau N, Dobos M, Zhang HP, Yang N, Ponzone A, Armarego WL, and Cotton RG. (1998) Dihydropteridine reductase deficiency: physical structure of the QDPR gene, identification of two new mutations and genotype-phenotype correlations Hum Mutat 12, 267–273. 128. Huang CY, Max EE, and Kaufman S. (1973) Purification and characterization of phenylalanine hydroxylase-stimulating protein from rat liver J Biol Chem 248, 4235–4241. 129. Davis MD, Kaufman S, and Milstien S. (1992) Distribution of 4a-hydroxytetrahydropterin dehydratase in rat tissues. Comparison with the aromatic amino acid hydroxylases FEBS Lett 302, 7376. 538 130. Schallreuter KU, Wood JM, Ziegler I, Lemke KR, Pittelkow MR, Lindsey NJ, and Gutlich M. (1994) Defective tetrahydrobiopterin and catecholamine biosynthesis in the depigmentation disorder vitiligo Biochim Biophys Acta 1226, 181–192. 131. Lei XD, Woodworth CD, Johnen G, and Kaufman S. (1997) Expression of 4alphacarbinolamine dehydratase in human epidermal keratinocytes Biochem Biophys Res Commun 238, 556–559. 132. Schallreuter KU, Moore J, Wood JM, Beazley WD, Peters EM, Marles LK, BehrensWilliams SC, Dummer R, Blau N, and Thöny B. (2001) Epidermal H(2)O(2) accumulation alters tetrahydrobiopterin (6BH4) recycling in vitiligo: identification of a general mechanism in regulation of all 6BH4-dependent processes? J Invest Dermatol 116, 167–174. 133. Lei XD, and Kaufman S. (1998) Human white blood cells and hair follicles are good sources of mRNA for the pterin carbinolamine dehydratase/dimerization cofactor of HNF1 for mutation detection Biochem Biophys Res Commun 248, 432–435. 134. Eskinazi R, Thony B, Svoboda M, Robberecht P, Dassesse D, Heizmann CW, Van Laethem JL, and Resibois A. (1999) Overexpression of pterin-4a-carbinolamine dehydratase/dimerization cofactor of hepatocyte nuclear factor 1 in human colon cancer Am J Pathol 155, 1105–1113. 135. von Strandmann EP, Senkel S, Ryffel G, and Hengge UR. (2001) Dimerization cofactor of hepatocyte nuclear factor 1/pterin-4alpha-carbinolamine dehydratase is necessary for pigmentation in Xenopus and overexpressed in primary human melanoma lesions Am J Pathol 158, 2021–2029. 136. Schallreuter KU, Wood JM, Pittelkow MR, Gütlich M, Lembke KR, Rödl W, Swanson NN, Hitzemann K, and Ziegler I. (1994) Regulation of melanin biosynthesis in the human epidermis by tetrahydrobiopterin Science 263, 1444–1446. 137. Strandmann EP, Senkel S, and Ryffel GU. (1998) The bifunctional protein DCoH/PCD, a transcription factor with a cytoplasmic enzymatic activity, is a maternal factor in the rat egg and expressed tissue specifically during embryogenesis Int J Dev Biol 42, 53–59. 138. Resibois A, Cuvelier L, Svoboda M, Heizmann CW, and Thony B. (1999) Immunohistochemical localisation of pterin-4alpha-carbinolamine dehydratase in rat peripheral organs Histochem Cell Biol 111, 381–390. 139. Depaepe V, Cuvelier L, Thony B, and Resibois A. (2000) Pterin-4alpha-carbinolamine dehydratase in rat brain. I. Patterns of co-localization with tyrosine hydroxylase Brain Res Mol Brain Res 75, 76–88. 539 140. Thöny B, Neuheiser F, Kierat L, Rolland MO, Guibaud P, Schlüter T, Germann R, Heidenreich RA, Duran M, de Klerk JBC, Ayling JE, and Blau N. (1998) Mutations in the pterin-4a-carbinolamine dehydratase gene (PCBD) are causative for a benign form of hyperphenylalaninemia Hum Genet 103, 162–167. 141. Thöny B, Neuheiser F, Kierat L, Rolland MO, Guibaud P, Schluter T, Germann R, Heidenreich RA, Duran M, de Klerk JB, Ayling JE, and Blau N. (1998) Mutations in the pterin-4alpha-carbinolamine dehydratase (PCBD) gene cause a benign form of hyperphenylalaninemia Hum Genet 103, 162–167. 142. Thöny B, Neuheiser F, Kierat L, Blaskovics M, Arn PH, Ferreira P, Rebrin I, Ayling, J, and Blau N. (1998) Hyperphenylalaninemia with high levels of 7-biopterin is associated with mutations in the PCBD gene encoding the bifunctional protein pterin-4acarbinolamine dehydratase and transcriptional coactivator (DCoH) Am J Hum Genet 62, 1302–1311. 143. Pontoglio M, Barra J, Hadchouel M, Doyen A, Kress C, Bach JP, Babinet C, and Yaniv M. (1996) Hepatocyte nuclear factor 1 inactivation results in hepatic dysfunction, phenylketonuria, and renal Fanconi syndrome Cell 84, 575–585. 144. Lei XD, and Kaufman S. (1998) Identification of hepatic nuclear factor 1 binding sites in the 5’ flanking region of the human phenylalanine hydroxylase gene: implication of a dual function of phenylalanine hydroxylase stimulator in the phenylalanine hydroxylation system Proc Natl Acad Sci USA 95, 1500–1504. 145. Kaufman S. (1991) Some metabolic relationships between biopterin and folate: implications for the “methyl trap hypothesis” Neurochem Res 16, 1031–1036. 146. Craine JE, Hall ES, and Kaufman S. (1972) The isolation and characterization of dihydropteridine reductase from sheep liver J Biol Chem 247, 6082–6091. 147. Firgaira FA, Cotton RG, and Danks DM. (1981) Isolation and characterization of dihydropteridine reductase from human liver Biochem J 197, 31–43. 148. Kappock TJ, and Caradonna JP. (1996) Pterin-dependent amino acid hydroxylases Chem Rev 96, 2659–2756. 149. Taguchi, H, and Armarego WL. (1998) Glyceryl-ether monooxygenase [EC 1.14.16.5]. A microsomal enzyme of ether lipid metabolism Med Res Rev 18, 43–89. 150. Tietz A, Lindberg M, and Kennedy EP. (1964) A new pteridine-requiring enzyme system for the oxidation of glyceryl ethers J Biol Chem 239, 4081–4090. 151. Kaufman S. (1957) The enzymatic conversion of phenylalanine to tyrosine J Biol Chem 226, 511–524. 152. Kaufman S. (1971) The phenylalanine hydroxylating system from mammalian liver Adv Enzymol Relat Areas Mol Biol 35, 245–319. 540 153. Shiman R, Jones SH, and Gray DW. (1990) Mechanism of phenylalanine regulation of phenylalanine hydroxylase J Biol Chem 265, 11633–11642. 154. Lazarus RA, Wallick DE, Dietrich RF, Gottschall DW, Benkovic SJ, Gaffney BJ, and Shiman R. (1982) The mechanism of phenylalanine hydroxylase Fed Proc 41, 2605–2607. 155. Dix TA, Kuhn DM, and Benkovic SJ. (1987) Mechanism of oxygen activation by tyrosine hydroxylase Biochem 26, 335433–61. 156. Haavik J, and Flatmark T. (1987) Isolation and characterization of tetrahydropterin oxidation products generated in the tyrosine 3-monooxygenase (tyrosine hydroxylase) reaction Eur J Biochem 168, 21–26. 157. Hufton SE, Jennings IG, and Cotton RG. (1995) Structure and function of the aromatic amino acid hydroxylases Biochem J 311 (Pt 2), 353–366. 158. Mitnaul LJ, and Shiman R. (1995) Coordinate regulation of tetrahydrobiopterin turnover and phenylalanine hydroxylase activity in rat liver cells Proc Natl Acad Sci USA 92, 885–889. 159. Pey AL, Perez B, Desviat LR, Martinez MA, Aguado C, Erlandsen H, Gamez A, Stevens RC, Thorolfsson M, Ugarte M, and Martinez A. (2004) Mechanisms underlying responsiveness to tetrahydrobiopterin in mild phenylketonuria mutations Hum Mutat 24, 388–399. 160. Thöny B, Ding Z, and Martinez A. (2004) Tetrahydrobiopterin protects phenylalanine hydroxylase activity in vivo: Implications for tetrahydrobiopterin-responsive hyperphenylalaninemia FEBS Lett 577, 507–511. 161. Milstien S, and Kaufman S. (1975) Studies on the phenylalanine hydroxylase system in liver slices J Biol Chem 250, 4777–4781. 162. Fukushima T, and Nixon JC. (1980) Analysis of reduced forms of biopterin in biological tissues and fluids Anal Biochem 102, 176–188. 163. Teigen K, and Martínez A. (2003) Probing cofactor specificity in phenylalanine hydroxylase by molecular dynamics simulations J Biomol Struct Dyn 20, 733–740. 164. Kure S, Sato K, Fujii K, Aoki Y, Suzuki Y, Kato S, and Matsubara Y. (2004) Wild-type phenylalanine hydroxylase activity is enhanced by tetrahydrobiopterin supplementation in vivo: an implication for therapeutic basis of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency Mol Genet Metab 83, 150–156. 165. Almas B, Le Bourdelles B, Flatmark T, Mallet J, and Haavik J. (1992) Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation. Structure/activity studies and mechanistic implications Eur J Biochem 209, 249–255. 541 166. Wei CC, Crane BR, and Stuehr DJ. (2003) Tetrahydrobiopterin radical enzymology Chem Rev 103, 2365–2383. 167. Stuehr DJ, Santolini J, Wang ZQ, Wei CC, and Adak S. (2004) Update on mechanism and catalytic regulation in the NO synthases J Biol Chem 279, 36167–36170. 168. Giovanelli J, Campos KL, and Kaufman S. (1991) Tetrahydrobiopterin, a cofactor for rat cerebellar nitric oxide synthase, does not function as a reactant in the oxygenation of arginine Proc Natl Acad Sci USA 88, 7091–7095. 169. Pastor CM, Williams D, Yoneyama T, Hatakeyama K, Singleton S, Naylor E, and Billiar TR. (1996) Competition for tetrahydrobiopterin between phenylalanine hydroxylase and nitric oxide synthase in rat liver J Biol Chem 271, 24534–24538. 170. Boadle-Biber MC. (1993) Regulation of serotonin synthesis Prog Biophys Mol Biol 60, 1–15. 171. Alp NJ, Mussa S, Khoo J, Cai S, Guzik T, Jefferson A, Goh N, Rockett KA, and Channon KM. (2003) Tetrahydrobiopterin-dependent preservation of nitric oxidemediated endothelial function in diabetes by targeted transgenic GTP-cyclohydrolase I overexpression J Clin Invest 112, 725–735. 172. Pannirselvam M, Simon V, Verma S, Anderson T, and Triggle CR. (2003) Chronic oral supplementation with sepiapterin prevents endothelial dysfunction and oxidative stress in small mesenteric arteries from diabetic (db/db) mice Br J Pharmacol 140, 701–706. 173. Ihlemann N, Rask-Madsen C, Perner A, Dominguez H, Hermann T, Kober L, and Torp-Pedersen C. (2003) Tetrahydrobiopterin restores endothelial dysfunction induced by an oral glucose challenge in healthy subjects Am J Physiol Heart Circ Physiol 285, H875–882. 174. Meininger CJ, Cai S, Parker JL, Channon KM, Kelly KA, Becker EJ, Wood MK, Wade LA, and Wu G. (2004) GTP cyclohydrolase I gene transfer reverses tetrahydrobiopterin deficiency and increases nitric oxide synthesis in endothelial cells and isolated vessels from diabetic rats Faseb J 18, 1900–1902. 175. Delgado-Esteban M, Almeida A, and Medina JM. (2002) Tetrahydrobiopterin deficiency increases neuronal vulnerability to hypoxia J Neurochem 82, 1148–1159. 176. Alp NJ, and Channon KM. (2004) Regulation of endothelial nitric oxide synthase by tetrahydrobiopterin in vascular disease Arterioscler Thromb Vasc Biol 24, 413–420. 177. Kirsch M, Korth HG, Stenert V, Sustmann R, and de Groot H. (2003) The autoxidation of tetrahydrobiopterin revisited. Proof of superoxide formation from reaction of tetrahydrobiopterin with molecular oxygen J Biol Chem 278, 24481–24490. 542 178. Blau N, and Erlandsen H. (2004) The metabolic and molecular bases of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency Mol Genet Metab 82, 101–111. 179. Tanaka K, Kaufman S, and Milstien S. (1989) Tetrahydrobiopterin, the cofactor for aromatic amino acid hydroxylases, is synthesized by and regulates proliferation of erythroid cells Proc Natl Acad Sci U S A 86, 5864–5867. 180. Anastasiadis PZ, Bezin L, Imerman BA, Kuhn DM, Louie MC, and Levine RA. (1997) Tetrahydrobiopterin as a mediator of PC12 cell proliferation induced by EGF and NGF Eur J Neurosci 9, 1831–1837. 181. Tanaka J, Koshimura K, Murakami Y, and Kato Y. (2002) Possible involvement of tetrahydrobiopterin in the trophic effect of insulin-like growth factor-1 on rat pheochromocytoma-12 (PC12) cells Neurosci Lett 328, 201–203. 182. Koshimura K, Murakami Y, Tanaka J, and Kato Y. (1998) Self-protection of PC12 cells by 6R-tetrahydrobiopterin from nitric oxide toxicity J Neurosci Res 54, 664–672. 183. Kojima S, Ona S, Iizuka I, Arai T, Mori H, and Kubota K. (1995) Antioxidative activity of 5,6,7,8-tetrahydrobiopterin and its inhibitory effect on paraquat-induced cell toxicity in cultured rat hepatocytes Free Radic Res 23, 419–430. 184. Shimizu S, Ishii M, Kawakami Y, Momose K, and Yamamoto T. (1998) Protective effects of tetrahydrobiopterin against nitric oxide-induced endothelial cell death Life Sci 63, 1585–1592. 185. Cho S, Volpe BT, Bae Y, Hwang O, Choi HJ, Gal J, Park LC, Chu CK, Du J, and Joh TH. (1999) Blockade of tetrahydrobiopterin synthesis protects neurons after transient forebrain ischemia in rat: a novel role for the cofactor J Neurosci 19, 878–889. 186. Hyland K, and Munk-Martin TL. (2001) Tetrahydrobiopterin regulates tyrosine hydroxylase and phenylalanine hydroxylase gene expression in dominantly inherited GTP cyclohydrolase deficiency J Inherit Metab Dis 24 (supplement), 30. 187. Linscheid P, Schaffner A, and Schoedon G. (1998) Modulation of inducible nitric oxide synthase mRNA stability by tetrahydrobiopterin in vascular smooth muscle cells Biochem Biophys Res Commun 243, 137–141. 188. Desviat LR, Perez B, Belanger-Quintana A, Castro M, Aguado C, Sanchez A, Garcia MJ, Martinez-Pardo M, Ugarte M. (2004) Tetrahydrobiopterin responsiveness: results of the BH4 loading test in 31 Spanish PKU patients and correlation with their genotype Mol Genet Metab 83, 157–162. 189. Koshimura K, Miwa S, Lee K, Fujiwara M, and Watanabe Y. (1990) Enhancement of dopamine release in vivo from the rat striatum by dialytic perfusion of 6R-L-erythro5,6,7,8-tetrahydrobiopterin J Neurochem 54, 1391–1397. 543 190. Mataga N, Imamura K, and Watanabe Y. (1991) 6R-tetrahydrobiopterin perfusion enhances dopamine, serotonin, and glutamate outputs in dialysate from rat striatum and frontal cortex Brain Res 551, 64–71. 191. Koshimura K, Miwa S, and Watanabe Y. (1994) Dopamine-releasing action of 6R-Lerythro-tetrahydrobiopterin: analysis of its action site using sepiapterin J Neurochem 63, 649–654. 192. Liang LP, and Kaufman S. (1998) The regulation of dopamine release from striatum slices by tetrahydrobiopterin and L-arginine-derived nitric oxide Brain Res 800, 181–186. 193. Smith I, and Wolff OH. (1974) Natural history of phenylketonuria and influence of early treatment Lancet 2, 540–544. 194. Blau N, Thöny B, Cotton RGH, and Hyland K (2001) in: The Metabolic and Molecular Bases of Inherited Disease, 8th ed, pp. 1725–1776 (Scriver CR, Beaudet AL, Sly WS, Valle, D, and Vogelstein B, Eds.) McGraw-Hill, New York. 195. Segawa M, Hosaka A, Miyagawa F, Nomura Y, and Imai H. (1976) Hereditary progressive dystonia with marked diurnal fluctuation Adv Neurol 14, 215–233. 196. Nygaard TG, Trugman JM, de Yebenes JG, and Fahn S. (1990) Dopa-responsive dystonia: the spectrum of clinical manifestations in a large North American family Neurology 40, 66–69. 197. Ichinose H, Ohye T, Takahashi E, Seki N, Hori T, Segawa M, Nomura Y, Endo K, Tanaka H, Tsuji S. et al. (1994) Hereditary progressive dystonia with marked diurnal fluctuation caused by mutations in the GTP cyclohydrolase I gene Nat Genet 8, 236–242. 198. Furukawa Y, and Kish SJ. (1999) Dopa-responsive dystonia: recent advances and remaining issues to be addressed Mov Disord 14, 709–715. 199. Ichinose H, Suzuki T, Inagaki H, Ohye T, and Nagatsu T. (1999) Molecular genetics of dopa-responsive dystonia Biol Chem 380, 1355–1364. 200. Curtius HC, Niederwieser A, Levine R, and Muldner H. (1984) Therapeutic efficacy of tetrahydrobiopterin in Parkinson’s disease Adv Neurol 40, 463–466. 201. Tani Y, Fernell E, Watanabe Y, Kanai T, and Langstrom B. (1994) Decrease in 6R5,6,7,8-tetrahydrobiopterin content in cerebrospinal fluid of autistic patients Neurosci Lett 181, 169–172. 202. Bottiglieri T, Hyland K, Laundy M, Godfrey P, Carney MW, Toone BK, and Reynolds EH. (1992) Folate deficiency, biopterin and monoamine metabolism in depression Psychol Med 22, 871–876. 544 203. Barford PA, Blair JA, Eggar C, Hamon C, Morar C, and Whitburn SB. (1984) Tetrahydrobiopterin metabolism in the temporal lobe of patients dying with senile dementia of Alzheimer type J Neurol Neurosurg Psychiatry 47, 736–738. 204. Curtius HC, Niederwieser A, Levine RA, Lovenberg W, Woggon B, and Angst J. (1983) Successful treatment of depression with tetrahydrobiopterin Lancet 1, 657–658. 205. Fernell E, Watanabe Y, Adolfsson I, Tani Y, Bergstrom M, Hartvig P, Lilja A, von Knorring AL, Gillberg C, and Langstrom B. (1997) Possible effects of tetrahydrobiopterin treatment in six children with autism–clinical and positron emission tomography data: a pilot study Dev Med Child Neurol 39, 313–318. 206. Schallreuter KU, Wood JM, Pittelkow MR, Gutlich M, Lemke KR, Rodl W, Swanson NN, Hitzemann K, and Ziegler I. (1994) Regulation of melanin biosynthesis in the human epidermis by tetrahydrobiopterin Science 263, 1444–1446. 207. Schallreuter KU, Beazley WD, Hibberts NA, Swanson NN, and Pittelkow MR. (1998) Perturbed epidermal pterin metabolism in Hermansky-Pudlak syndrome J Invest Dermatol 111, 511–516. 208. Schallreuter KU. (1999) A review of recent advances on the regulation of pigmentation in the human epidermis Cell Mol Biol (Noisy-le-grand) 45, 943–949. 209. Kure S, Hou DC, Ohura T, Iwamoto H, Suzuki S, Sugiyama N, Sakamoto O, Fujii K, Matsubara Y, and Narisawa K. (1999) Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency J Pediatr 135, 375–378. 210. Erlandsen H, Pey AL, Gamez A, Perez B, Desviat LR, Aguado C, Koch R, Surendran S, Tyring S, Matalon R, Scriver CR, Ugarte M, Martinez A, and Stevens RC. (2004) Correction of kinetic and stability defects by tetrahydrobiopterin in phenylketonuria patients with certain phenylalanine hydroxylase mutations Proc Natl Acad Sci USA 101, 16903–16908. 211. Bode VC, McDonald JD, Guenet JL, and Simon D. (1988) hph-1: a mouse mutant with hereditary hyperphenylalaninemia induced by ethylnitrosourea mutagenesis Genetics 118, 299–305. 212. Hyland K, Gunasekara RS, Munk-Martin TL, Arnold LA, and Engle T. (2003) The hph1 mouse: a model for dominantly inherited GTP-cyclohydrolase deficiency Ann Neurol 54 Suppl 6, S46–48. 213. Khoo JP, Nicoli T, Alp NJ, Fullerton J, Flint J, and Channon KM. (2004) Congenic mapping and genotyping of the tetrahydrobiopterin-deficient hph-1 mouse Mol Genet Metab 82, 251–254. 545 214. Sumi-Ichinose C, Urano F, Kuroda R, Ohye T, Kojima M, Tazawa M, Shiraishi H, Hagino Y, Nagatsu T, Nomura T, and Ichinose H. (2001) Catecholamines and serotonin are differently regulated by tetrahydrobiopterin. A study from 6-pyruvoyltetrahydropterin synthase knockout mice J Biol Chem 276, 41150–41160. 215. Elzaouk L, Leimbacher W, Turri M, Ledermann B, Bürki K, Blau N, and Thöny B. (2003) Dwarfism and low insulin-like growth factor-1 due to dopamine depletion in Pts-/- mice rescued by feeding neurotransmitter precursors and H4-biopterin J Biol Chem 278, 28303–28311. 216. Thompson JD, Higgins DG, and Gibson TJ. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res 22, 4673–4680. ADDRESS FOR CORRESPONDENCE: Beat Thöny Division of Clinical Chemistry and Biochemistry University Children’s Hospital Steinwiesstrasse 75 8032 Zurich Switzerland Tel.: +411-266 7622 Fax: +411-266 7169 E-Mail: [email protected] 546