Download Tetrahydrobiopterin and its functions

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.
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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.
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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
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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.
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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.
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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).
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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-
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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
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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].
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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
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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
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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
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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
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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].
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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
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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).
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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.
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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.
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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]
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