Download Structure, mechanism and function of prenyltransferases

Eur. J. Biochem. 269, 3339–3354 (2002) FEBS 2002
doi:10.1046/j.1432-1033.2002.03014.x
REVIEW ARTICLE
Structure, mechanism and function of prenyltransferases
Po-Huang Liang, Tzu-Ping Ko and Andrew H.-J. Wang
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
In this review, we summarize recent progress in studying
three main classes of prenyltransferases: (a) isoprenyl pyrophosphate synthases (IPPSs), which catalyze chain elongation of allylic pyrophosphate substrates via consecutive
condensation reactions with isopentenyl pyrophosphate
(IPP) to generate linear polymers with defined chain lengths;
(b) protein prenyltransferases, which catalyze the transfer of
an isoprenyl pyrophosphate (e.g. farnesyl pyrophosphate) to
a protein or a peptide; (c) prenyltransferases, which catalyze
the cyclization of isoprenyl pyrophosphates. The prenyltransferase products are widely distributed in nature and
serve a variety of important biological functions. The catalytic mechanism deduced from the 3D structure and other
biochemical studies of these prenyltransferases as well as
how the protein functions are related to their reaction
mechanism and structure are discussed. In the IPPS reaction,
we focus on the mechanism that controls product chain
length and the reaction kinetics of IPP condensation in the
cis-type and trans-type enzymes. For protein prenyltransferases, the structures of Ras farnesyltransferase and Rab
geranylgeranyltransferase are used to elucidate the reaction
mechanism of this group of enzymes. For the enzymes
involved in cyclic terpene biosynthesis, the structures and
mechanisms of squalene cyclase, 5-epi-aristolochene synthase, pentalenene synthase, and trichodiene synthase are
summarized.
Isoprenoids are an extensive group of natural products with
diverse structures consisting of various numbers of fivecarbon isopentenyl pyrophosphate (IPP) units [1,2]. The
more than 23 000 isoprenoid compounds identified thus far
serve a variety of essential biological functions in Eukarya,
Bacteria and Archaea. For example, steroids are cyclic
isoprenoids, which have distinct biological functions as
hormones [3]. Carotenoids contain highly conjugated
structures for absorption of light and are the most common
accessory pigments in all green plants and many photosynthetic bacteria [4]. Retinoids are involved in morphogenesis
and are the light-sensitive element in vision. Prenylated
proteins including Ras and other G-proteins are involved in
specific signal-transduction pathways [5,6].
Many linear isoprenoids, generally synthesized from C15
farnesyl pyrophosphate (FPP) and IPP, are found in nature
[7]. The enzymes responsible for the synthesis of linear
isoprenyl pyrophosphates can be classified as cis- and transisoprenyl pyrophosphate synthase (IPPS) according to the
stereochemical outcome of their products [8]. As shown in
Fig. 1, all-trans-geranyl pyrophosphate (GPP) and FPP are
synthesized by trans-type geranyl pyrophosphate synthase
(GPPS) and farnesyl pyrophosphate synthase (FPPS),
respectively. Starting from FPP, a variety of differentchain-length products are generated by the corresponding
synthases. The geranylgeranyl pyrophosphate synthase
(GGPPS) and farnesylgeranyl pyrophosphate synthase
(FGPPS) produce C20 and C25 all-trans-polyprenyl pyrophosphates to make C20–C20 and C20-C25 ether-linked lipids
in archeon [9–11]. The C40 product of octaprenyl pyrophosphate synthase (OPPS) constitutes the side chain of
ubiquinone in Escherichia coli [12–14]. Several cis-isoprenyl
Keywords: chain elongation; isoprenoid; lipid carrier;
prenyltransferase; site-directed mutagenesis; 3D structure.
Correspondence to P.-H. Liang, Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
Fax: +886 2 2788 9759, Tel.: +886 2 2785 5696 ext. 6070, E-mail: [email protected] or A.H.-J. Wang, Fax: +886 2788 2043,
E-mail: [email protected]
Abbreviations: FPP, farnesyl pyrophosphate; GPP, geranyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; IPP, isopentenyl pyrophosphate; UPP, undecaprenyl pyrophosphate; IPPS, isoprenyl pyrophosphate synthase; UPPS, undecaprenyl pyrophosphate synthase; DDPPS,
dehydrodolichyl pyrophosphate synthase; PPPS, polyprenyl pyrophosphate synthase; GPPS, geranyl pyrophosphate synthase; FPPS, farnesyl
pyrophosphate synthase; GGPPS, geranylgeranyl pyrophosphate synthase; FGPPS, farnesylgeranyl pyrophosphate synthase; HexPPS, hexaprenyl pyrophosphate synthase; HepPPS, heptaprenyl pyrophosphate synthase; OPPS, octaprenyl pyrophosphate synthase; SPPS, solanesyl
pyrophosphate synthase; DPPS, decaprenyl pyrophosphate synthase; FTase, farnesyltransferases; GGTase, geranylgeranyltransferase.
Enzymes: UPPS from Escherichia coli (EC 2.5.1.31); DDPPS from yeast Saccharomyces cerevisiae (EC 2.5.1.31); PPPS from Arabidopsis thaliana
(EC 2.5.1.31); FPPS from E. coli (EC 2.5.1.10); GGPPS from yeast (EC 2.5.1.29); FGPPS from Aeropyrum pernix (EC 2.5.1.33); HexPPS from
Bacillus stearothermophilus and yeast (EC 2.5.1.30); HepPPS from Mycobacterium tuberculosis (EC 2.5.1.30); OPPS from E. coli (EC 2.5.1.11);
SPPS from Rhodobacter capsulatus (EC 2.5.1.11); DDPPS from human (EC 2.5.1.31); Ras farnesyltransferase from human (EC 2.1.1.100); Rab
geranylgeranyltransferase from human (EC 2.1.1.100); squalene cyclase from Alicyclobacillus acidocaldarius (2.5.1.31); 5-epi-aristolochene synthase
from Tobacco (EC 4.2.3.6); pentalenene synthase from Streptomyces UC5319 (EC 4.2.3.7); trichodiene synthase from Fusarium sporotrichioides
(EC 4.2.3.6).
(Received 22 March 2002, accepted 17 May 2002)
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3340 P.-H. Liang et al. (Eur. J. Biochem. 269)
Fig. 1. Synthesis of linear all trans-isoprenyl
pyrophosphates (top) and trans,cis-isoprenyl
pyrophosphates (bottom) catalyzed by transIPPSs and cis-IPPSs, respectively. The
stereoisomers are not specified in the nomenclature of the enzymes. Beside a trans-FPPS, a
cis-type FPPS from M. tuberculosis has been
shown to synthesize cis,trans-FPP.
pyrophosphate synthases including solanesyl pyrophosphate synthase (SPPS) [15–17], decaprenyl pyrophosphate
synthase (DPPS) [18], heptaprenyl pyrophosphate synthase
(HepPPS) [19], and hexaprenyl pyrophosphate synthase
(HexPPS) [20,21] are responsible for making C45, C50, C35,
and C30 side chains, respectively, of ubiquinone in different
species [22].
Among the cis-polyprenyl pyrophosphates, the C55
product of the bacterial undecaprenyl pyrophosphate
synthase (UPPS) serves as a lipid carrier in cell wall
peptidoglycan biosynthesis [23,24]. Its homologous dehydrodolichyl pyrophosphate synthase (DDPPS) in eukaryotes is responsible for making C55–C100 dolichols for
glycoprotein biosynthesis, a pathway similar to that of the
bacterial peptidoglycan synthesis [25,26]. An even longer
C120 polymer was found as the final product of an isoprenyl
pyrophosphate synthase in plant Arabidopsis thaliana with
unknown function [27]. As the cis-prenyltransferases commonly synthesize long-chain products, a unique short-chain
cis,trans-FPP is made by Mycobacterium tuberculosis FPPS
which utilizes C10 GPP and IPP to produce a FPP with a cis
double bond [28] (Fig. 1). This bacterium has a decaprenyl
pyrophosphate synthase (DPPS) to produce C50 decaprenyl
pyrophosphate as a lipid carrier, which is one IPP unit
shorter than UPP found in other bacteria.
The products of these prenyltransferases have specific
chain lengths essential for their biological functions. An
intriguing question is how do they achieve product chainlength specificity. In theory, restriction of the size of the
enzyme active site should play a major role in determining
the chain length of the final product. With the increasing
numbers of 3D structures available for prenyltransferases,
the mechanism of product chain-length determination has
begun to be elucidated [29]. The cis-type UPPS crystal
structures from Micrococcus luteus and E. coli have been
solved by Fujihashi et al. [30] and Ko et al. [31], respectively, providing the first two structures of the cis-prenyltransferase family. The structure in conjunction with
site-directed mutagenesis studies has revealed how
cis-prenyltransferase controls its product size [31].
Protein prenyltransferases form another prenyltransferase family. This enzyme catalyzes the transfer of the
carbon moiety of FPP or GGPP to a conserved cysteine
residue in a CaaX motif of protein and peptide substrates
[32]. This postranslational modification is essential for a
number of proteins including Ras, Rab, nuclear lamins,
trimeric G-protein c subunits, protein kinases, and small
Ras-related GTP-binding proteins [33,34]. The addition of a
farnesyl group to these proteins is required to anchor them
to the cell membrane, a step required for them to function.
As oncogenic forms of Ras in nearly 30% of human cancers
were observed, inhibition of Ras farnesyltransferases
(FTases) became a new strategy for anticancer therapy
[35,36]. The crystal structure of a mammalian FTase had
been determined at 2.25 Å resolution [37]. Subsequently, the
structures of the enzyme in complex with FPP substrate [38]
and a ternary complex of the enzyme with FPP and a CaaX
peptide substrate were solved [39]. Later, a Rab geranylgeranyltransferase (GGTase) was solved at 2.0 Å resolution
[40].
FPP is considered to be a branching point in the synthesis
of different types of natural isoprenoids. A number of
enzymes catalyze cyclization of FPP to generate natural
products such as pentalenene (pentalenene synthase), 5-epiaristolochene (5-epi-aristolochene synthase) and trichodiene
(trichodiene synthase). Squalene synthase catalyzes the
cyclization of squalene which is synthesized by the coupling
of two FPP molecules. The crystal structures of these
enzymes have been solved and provide insights into the
catalytic mechanism of terpenoid cyclization [41–44].
This review summarizes these recent advances in the
structural and mechanistic studies of the above three
families of prenyltransferases with emphasis on IPPS, which
has essential biological functions (Table 1). A general
mechanism of product chain-length determination and the
reaction kinetics derived from a pre-steady-state kinetic
analysis for trans-IPPS and cis-IPPS are described.
CLASS I: ISOPRENYL PYROPHOSPHATE
SYNTHASES (IPPSs)
Structure and active site of trans-IPPS
Over the past decade, many trans-IPPSs have been purified
and their genes cloned [45,46]. The deduced amino-acid
sequences of these enzymes show amino-acid sequence
homology and two common DDxxD motifs [47], suggesting
that they evolved from the same origin (Fig. 2) [48,49].
These Asp-rich motifs were recognized from the 3D
structure [50] and site-directed mutagenesis studies [51–56]
to be involved in substrate binding and catalysis via
chelation with Mg2+, a cofactor required for enzyme
activity.
The first crystal structure of a trans-prenyltransferase
reported is that of avian FPPS [50]. As shown in Fig. 3A,
this 2.6-Å-resolution structure contains 13 a helices, 10 of
them surrounding the large central cavity. Two conserved
DDxxD sequences are located in this deep cleft, which
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7 Table 1. The biological functions and three-dimensional structures of prenyltransferases presented in this review.
Prenyltransferases
Biological functions
3D structure [ref]
trans-FPPS
Precursor of steroids, cholesterol, sesquiterpenes, farnesylated proteins,
heme, and vitamin K12
Precursor of carotenoids, retinoids, diterpenes, geranylgeranylated
chlorophylls, and archaeal ether linked lipids
Archaeal ether linked lipids
Ubiquinone side chains
[50,57]
Lipid carrier for peptidoglycan synthesis
Lipid carrier for glycoprotein synthesis
Farnesylated Ras for signal transduction
Geranylgeranylated Rab for signal transduction
Precursor of cholesterol
Precursor of antifungal phytoalexin capsidol
Precursor of pentalenolactone antibiotics
Precursor of antibiotics and mycotoxins
[30,31]
trans-GGPPS
trans-GFPPS
trans-HexPPS
–DPPS
cis-UPPS
cis-DDPPS
Ras FTase
Rab GGTase
Squalene cyclase
epi-Aristolochene synthase
Pentalenene synthase
Trichodiene synthase
forms the substrate-binding pocket. In site-directed mutagenesis studies of yeast FPPS, substitution of Asp with Ala
in the first negatively charged DDxxD motif decreased kcat
by 4–5 orders of magnitude [53]. In the second DDxxD
motif of yeast FPPS, the first and second Asp to Ala
Fig. 2. Sequence alignment of trans-prenyltransferases. The sequence-related proteins
including FPPS from E. coli, GGPPS from
yeast, FGPPS from E. coli, HexPPS
from yeast, HepPPS from B. subtilis, OPPS
from E. coli, SPPS from M. luteus, and DPPS
from human are shown. Black and gray
outlines indicate identical and similar amino
acid residues, respectively. The two DDxxD
motifs are conserved in all proteins. The 5th
amino acid upstream from the first DDxxD is
a large residue (Leu, Phe, or Tyr) for FPPS
and GGPPS, and is small amino acid (Ala) for
FGPPS, HexPPS, HepPPS, OPPS, SPPS, and
DPPS.
[37–39]
[40]
[41]
[42]
[43]
[44]
mutations resulted in kcat values 4–5 orders of magnitude
smaller [53]. However, when the third Asp was mutated to
Ala, there was a less pronounced effect ( 100-fold
smaller kcat) [53]. A 104)105 lower activity was observed
for the FPPS from Bacillus stearothermophilus when the first
3342 P.-H. Liang et al. (Eur. J. Biochem. 269)
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Fig. 3. (A) Crystal structure of trans-type FPPS and (B) schematic representation of the active site with FPP (product) and IPP bound. (A) The model
of avian FPPS is shown using a ribbon diagram. Two identical subunits are associated into a dimer by forming a four-layer helix bundle. The
N-terminal a helical hairpins in the outer layers are shown in blue and red for the two individual subunits, while the eight helices in the inner layers
are shown in cyan and magenta. Two small peripheral domains are colored green and yellow. The locations of the active sites are indicated by red
arrows, where the aspartate side chains of the two DDxxD motifs in each subunits are also shown in red. The phenylalanine side chains of F112 and
F113, which are involved in product length control, are shown in green. (B) The 5th amino acid (shown as a filled black circle) before the first
DDxxD motif in the sequence is located next to the tail of FPP. The large amino acid at this position can block further chain elongation of the
product and represents a mechanism to control the product chain length.
and second Asp of the second DDxxD motif were replaced
with Ala [55]. In contrast, the third Asp seems to be less
important to catalysis as its mutation to Ala only resulted in
6–16 times lower kcat values, but with a 10-fold increased
IPP Km value [55].
For rat FPPS, replacement of the second and third Asp
with Glu in the first Asp-rich motif decreased kcat by
1000-fold [52]. However, no significant change in the Km
values for IPP and GPP were observed. In the second
DDxxD motif of rat FPPS, substituting the first Asp with
Glu decreased kcat 90-fold and increased IPP Km 26-fold,
whereas GPP Km remained unchanged [51]. On the other
hand, mutation of the third Asp resulted in no change in the
kinetic parameters.
These results indicate that all the Asp residues in the two
DDxxD motifs except the last one in the second motif are
important for catalysis. In addition, the second motif is
essential for IPP binding. These results are consistent with
the cocrystal structure of avian FPP in complex with GPP
and IPP [57]. The structure of FPPS clearly shows that the
first DDxxD is bound to the allylic substrate GPP and the
second motif is the binding site of the homoallylic substrate
IPP. Site-directed mutagenesis of other amino acids around
the two DDxxD motifs was also performed to show their
effects in substrate binding and catalysis [54,56].
Amino-acid residues essential to product chain-length
determination of trans-prenyltransferases
In parallel with the site-directed mutagenesis studies, a
random chemical mutagenesis approach was used to select
FPPS mutants induced by NaNO2 treatment that could
synthesize longer-chain products. FPPS was converted into
GGPPS which synthesizes a C20 product by generating
mutations at position 81, 34 and 157 of FPPS from
B. stearothermophilus [58]. The Y81H mutant was the most
effective at increasing the production of GGPP (C20)
compared with FPP (C15) [58]. The subsequent site-directed
mutagenesis study in which Y81 was systematically replaced
with each of the other 19 amino-acid residues showed that
Y81A, Y81G and Y81S are capable of making hexaprenyl
pyrophosphate (C30) [59]. The final products of Y81C,
Y81H, Y81I, Y81L, Y81N, Y81T and Y81V are C25
geranylfarnesyl pyrophosphate. On the other hand, Y81D,
Y81E, Y81F, Y81K, Y81M, Y81Q, and Y81R cannot
synthesize products larger than GGPP (C20). It appears that
this residue, located at the fifth position before the first
DDxxD motif, is the key residue in determining product
chain length. Predicted from the secondary structure of
FPPS, this residue is located at a distance of 12 Å from
the first Asp-rich motif, which is similar to the length of the
hydrocarbon moiety of FPP [59]. The chain length of the
product catalyzed by these mutants is inversely proportional
to the accessible surface volume of the substituted aminoacid residue in the first DDxxD. Also in archaebacterial
GGPPS, mutation of Phe77, which is upstream from the
first DDxxD, led to a change in product [60]. For instance,
replacement of this large residue with the smaller Ser
resulted in the production of C25 rather than C20.
There was a similar finding in avian FPPS, in which
replacement of aromatic Phe112 and Phe113 with smaller
amino acids resulted in the product specificity shifting from
1 C15 (FPP) to C20 (GGPP) (F112A), C25 geranylfarnesyl
pyrophosphate (F113S) and longer products (F112A/
F113S double mutant) [57]. These two residues are located
in the fifth and sixth position before the first DDxxD
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motif. The X-ray crystal structure of the enzyme reveals
that the first DDxxD is near a large hydrophobic pocket
which contains the mutated Phe112 and Phe113 residues
(Fig. 3A). It is proposed that this pocket binds the growing
hydrocarbon tail of the product, and the first DDxxD is
responsible for the binding of its pyrophosphate moiety.
An enlarged active site of the mutant enzymes is probably
related to increased product size based on the 3D
structures of the FPPS in the apo state and to the allylic
substrate bound. Accordingly, an increase of 5.8 Å in the
depth of the hydrophobic pocket agrees with the 5.2 Å
difference of an extra IPP unit between FPP (C15) and
GGPP (C20). The role of F113 in controlling the product
chain length is further supported by the fact that GGPPS
from Methanobacterium thermoautotrophicum contains
phenylalanine and serine at the positions corresponding
to Phe112 and Phe113 in avian FPPS. From the X-ray
crystal structure and the sequence alignment of a variety of
polyprenyl pyrophosphate synthases (PPPSs), which generate C15, C20 and larger products, the importance of
Phe112 and Phe113 in the mechanism of product chainlength determination is evident. In conclusion, the large
amino-acid residue located before the first DDxxD motif
provides the ÔfloorÕ to block further elongation of the
product (Fig. 3B). Once this residue is replaced with a
smaller one, elongation can continue.
In addition to the 5th and 6th amino-acid residues, the
roles of the 8th and/or the 11th positions before the first
DDxxD in the control of product specificity of archaeal
GGPPS and FPPS were also examined [61]. The single
mutant (F77S, 5th amino acid residue before the first Asprich motif), double mutant (L74G/F77G) and triple mutant
(I71G/74G/F77G) of GGPPS mainly produce C25, C35 and
C40, respectively [61]. FPPS mutants display a similar
pattern. This indicates that replacing amino acids near the
5th amino acid with smaller ones can further increase
production of longer polymers.
Conversely, replacement of a small amino acid with a
larger one shortens the chain length of the product. The
avian FPPS mutants A116W and N114W produce C10 GPP
instead of C15 FPP synthesized by the wild-type enzyme [62].
These residues are also located in the hydrophobic pocket of
the allylic substrate site, as revealed by docking dimethylallyl pyrophosphate into the crystal structure of FPPS.
Dimethylallyl pyrophosphate is an isomer of IPP and its
condensation with IPP produces GPP. The mutations
A116W and N114W could fill in the bottom of the activesite cavity, forcing the synthesis of shorter GPPs. Steadystate kinetic measurements indicate that the two mutant
enzymes have larger kcat values with dimethylallyl pyrophosphate as substrate and binding of GPP is therefore
shifted to the allylic site because of the smaller internal space
of the active site.
Structure and active site of cis-IPPS
UPPS has been partially purified from Lactobacillus
plantarum and characterized [63]. Subsequently, the UPPS
encoding gene cloned from M. luteus is the first cisprenyltransferase gene identified, and the deduced aminoacid sequence shows no sequence similarity to those of
trans-prenyltransferases [64]. The sequence comparison
with UPPS allows the identification of many cis-type
IPPSs in bacteria, plant and animals as a family [65].
Several regions of conserved sequences can be identified
(Fig. 4). Broadly grouped, they are (with the amino-acid
sequence shown in parentheses): region I (20–32), region II
(42–46), region III (66–88), region IV (142–154) and region
V (190–224). Most of the fully conserved amino acids are
involved in catalysis, substrate binding, or structural
interactions as revealed later by the crystal structures and
mutagenesis studies.
Unlike the trans-type enzymes, cis-prenyltransferases
lack the DDxxD motifs, although they require Mg2+ for
activity. Earlier site-directed mutagenesis studies examined
the conserved Asp and Glu of E. coli UPPS and revealed
the importance of Asp26, Asp150 and Glu213 in substrate
binding and catalysis [66]. Replacement of Asp26 with Ala
results in 103-fold smaller kcat without any significant
change in FPP and IPP Km values. Mutagenesis of Asp150
to alanine leads to a 50 times larger IPP Km but no change
in FPP Km and kcat values. If Glu213 is replaced with Ala,
there is a 70-fold increase in IPP Km and a 100-fold
decrease in kcat. The subsequent 3D structure of UPPS
from M. luteus suggests that Asp29 (equivalent to Asp26
in E. coli UPPS) is located in a p-loop, a conserved motif
for the pyrophosphate-binding site in many phosphatebinding proteins such as nucleotide triphosphate hydrolase,
phosphofructokinase, cAMP-binding domain, and sugar
phosphatase [67]. Site-directed mutagenesis studies on
M. luteus UPPS have also identified Glu216 (equivalent
to Glu213 in the E. coli enzyme) as being responsible for
IPP binding via Mg2+ [68]. A hydrophobic cleft in the
enzyme with four positively charged Arg residues located
at the entrance of the cleft and the hydrophobic residues
covering the interior is proposed as the site for substrate
recognition [30].
The E. coli UPP structure reveals a larger hydrophobic
tunnel surrounded by two a helices (a2 and a3) and four
b strands (bB, bA, bD, and bC) as shown in Fig. 5A. In
contrast with the symmetric structure of M. luteus UPPS,
two protein conformations (open and closed forms) are
seen in the two subunits of E. coli UPPS, implicating a
closed/open conformational change mechanism in substrate binding and product release [31]. The difference
between the two conformers is mainly in the position of
the a3 helix. On the basis of site-directed mutagenesis
studies [31], the flexible loop with amino acids 72–83
connected to the a3 helix has been suggested to serve as a
hinge for the interconversion of two conformers. This
study also suggested a role for a Trp residue in the loop for
FPP binding and catalysis [69]. Fluorescent stopped-flow
technology and steady-state spectrophotometer have
recently been used to directly observe a Trp fluorescence
intensity change due to the change in protein conformation
during catalysis [70]. When Trp91, which is located in the
a3 helix, is mutated to Phe, the fluorescence quenching
upon addition of FPP is abolished, suggesting that the a3
helix moves toward the active site during substrate binding
[70]. Thus the change in UPPS conformation to a closed
form results in better interaction between the enzyme and
the substrates and intermediates. After the reaction, the
UPPS structure shifts to an open form for product release,
triggered by crowding of the prenyl chain of the product
because the large amino-acid residues seal the bottom of
the tunnel-shaped active site.
3344 P.-H. Liang et al. (Eur. J. Biochem. 269)
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Fig. 4. Alignment of the cis-type IPPSs. These
include, in turn, E. coli UPPS, yeast DDPPS
Rer2, Yeast DDPPS Srt1, M. tuberculosis FPs
Rv1086, M. tuberculosis DPPS Rv2361c,
Arabidopsis thaliana PPPS and human
DDPPS. The numbers and secondary-structural elements shown above the sequences
are for the UPPS from E. coli, based on
PROCHECK analysis of the crystal structure.
The green arrows denote the locations of
b-strands, and the cylinders in red, magenta
and cyan are for a helices, 310 helices and
turns, respectively. In the aligned sequences,
several conserved regions including (I) residues 20–32, (II) 42–46, (III) 66–88, (IV)
142–154, and (V) 190–224 can be identified,
which probably also have corresponding
secondary structures in common.
Mechanism of product chain-length determination
in cis-IPPS
E. coli UPPS has a hydrophobic tunnel formed by two
a helices and four b strands, which is sufficiently large to
accommodate the whole UPP (Fig. 5A). Large amino acids
including I62, L137, V105, and H103 are located on the
bottom of the tunnel [31]. Among the mutants produced by
replacing these residues with the smaller Ala, L137A
synthesizes C70 and C75 as major products, which are longer
than the C55 product of the wild-type enzyme [31]. Ko et al.
[31] proposed that this residue plays an essential role in
determining the product chain length by blocking further
product elongation, analogous to the above amino-acid
residue located upstream from the first DDxxD motif in the
trans-type enzyme (Fig. 5B). This residue may represent a
common residue in the mechanism of product chain-length
determination among cis-prenyltransferases. From their
sequence homology, yeast Rer2, which synthesizes longerchain C70–C80 products, has an Ala residue at this position.
The single UPPS mutation, L137A, has converted UPPS
into DDPPS in terms of product specificity. V105 may also
play a role in blocking chain elongation, as its mutation
increases the proportion of C60, C65 and C70 in the absence
of Triton, but it is not as critical as L137.
All cis-prenyltransferases so far identified have products
of chain length at least C55, except a short-chain FPPsynthesizing enzyme recently identified from M. tuberculosis. When the amino-acid sequence of UPPS is compared
with that of the short-chain FPPS, the A69 and A143 in
E. coli UPPS correspond to the large L84 and V156 in
M. tuberculosis FPPS, respectively. Substitution of Leu for
A69 in E. coli UPPS indeed leads to production of a greater
amount of C30 intermediate which is longer lived, suggesting
that this residue interferes with the chain elongation of the
C30 product. From the structure, it is reasonable to assume
that A69 is located midway in FPP elongation to the C55
product. The conclusion derived from the changes in
product specificity in E. coli UPPS mutants needs to be
confirmed when the structures of the other cis-prenyltransferases are available.
Reaction mechanism and kinetics of trans-IPPS
and cis-IPPS
An ionization–condensation–elimination mechanism has
been proposed for the trans-prenyltransferase reaction. As
shown below in Fig. 9A, the carbocation resulting from the
dissection of the pyrophosphate group from GPP is
proposed as the intermediate during the FPPS-catalyzed
condensation reaction of IPP with allylic pyrophosphate
substrate [1]. This conclusion is based on the finding that the
enzyme, which normally catalyzes the addition of a GPP to
IPP, is able to catalyze the hydrolysis of GPP [71]. Hydrolysis
carried out in H218 O or with (1S)-[1-3H]GPP shows that the
C–O bond is broken, and the chirality of the C1 carbon of
GPP is inverted in the process. Furthermore, the trifluoromethyl substituent at the C3 position or the fluoro atom at
the C2 position of the allylic substrate destabilizes the
carbocation and retards the enzyme reaction [72].
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Structure, mechanism and function of prenyltransferases (Eur. J. Biochem. 269) 3345
Fig. 5. (A) Two orthogonal views of the ribbon representation of an E. coli UPPS dimer and (B) the proposed active site of the E. coli UPPS located in
a tunnel-like crevice surrounded by a2, a3, bD, bB, bA, and bC. (A) The top view is perpendicular to, and the bottom view is parallel to, the molecular
dyad axis. The seven a helices and six b strands are shown in red and green, respectively, for subunit A, and in magenta and cyan for subunit B, and
they are labelled separately. The blue arrows indicate locations of the active sites, each having a substrate-binding tunnel formed by helices a2, a3
and the central b-sheet. (B) The substrate site is located on the top of the hydrophobic tunnel with D26 and D213 playing a significant role in
substrate binding and catalysis. A69 is in the midle of FPP chain elongation to the product. The large amino acid L137 on the bottom of the tunnel
is essential for determination of product chain length by blocking further elongation of UPP.
A tertiary carbocation on the C3 carbon of the IPP part is
proposed to form during the 1¢-4 condensation. The aza
analogues with nitrogen replacing the cationic carbon to
mimic the transition state have previously been demonstrated to strongly inhibit enzymes in isoprenoid biosynthesis
where the carbocation transition state is presumably formed
during catalysis [73]. An aza analogue of the transition state,
3-azageranylgeranyl diphosphate, is also a potent inhibitor
of GGPPS [74,75].
In the elimination step, a hydrogen is removed from C2
of IPP with simultaneous formation of a C¼C double
bond between C2 and C3. The formation of the trans or
cis double bond during the IPPS reaction depends on the
spatial arrangement of the IPP relative to the elongating
oligoprenyl substrate. In the trans-prenyltransferases, the
allylic substrate is added to the si face of the double bond
of the IPP, and the pro-R proton is removed from the
methylene group next to the double bond, with the
concomitant formation of a new trans double bond [72].
The amino acid involved in the hydrogen removal of IPP
in prenyltransferase has not yet been reported. However,
several important amino acids, including the nucleophilic
His309 responsible for the removal of the pro-R hydrogen,
have been proposed from analysis of the crystal structure
of pentalenene synthase, which catalyzes cyclization of
FPP initiated by the cleavage of its pyrophosphate moiety
and formation of a carbocation intermediate (see below
for details) [43]. The active-site residues Phe77 and Asn219
have been implicated in the stabilization of the carbocation intermediate [43]. In UPPS, there are several
conserved hydrophilic amino acids in the vicinity of
Asp26, including Asn28, Arg30, His43, Phe70, Ser71,
Arg194 and Glu198. It is conceivable that some of them
may play roles analogous to those of His309, Phe77 and
Asn219 in pentalenene synthase. However, to form a cis
double bond in UPPS, the position of the requisite
nucleophile (possibly His43) will need to be close to the
pro-S proton of IPP.
The reaction mechanism of the cis-prenyltransferases is
less well understood. The hydrolysis of the allylic substrate
by cis-prenyltransferases has not been observed. Analogously to the two DDxxD motifs in the trans-prenyltransferases, site-directed mutagenesis studies of UPPS indicate
that Asp and Glu play a significant role in IPP binding and
3346 P.-H. Liang et al. (Eur. J. Biochem. 269)
FEBS 2002
activity probably through co-ordination with Mg2+. The
IPP condensation kinetics for UPPS, DDPPS (cis-type) and
OPPS (trans-type) had been measured for comparison of
IPP condensation catalyzed by cis-IPPS and trans-IPPS
[76–78]. These experiments were performed under the
enzyme single-turnover condition using a rapid-quench
instrument, because the slow release of the large hydrophobic product limits IPPS catalysis under steady-state conditions. The IPP condensation rate constant in the UPPS
reaction is similar to that of OPPS (2.5 s)1 vs. 2.0 s)1)
derived from the time course data simulated by the Kinsim
program (Fig. 6). This suggests that the chemical environment of the active site in the two types of enzyme may be
similar, despite the completely different primary sequence.
Moreover, Triton could facilitate product release and
increase the UPPS steady-state rate by 190-fold by switching
the rate-limiting step from product release to IPP condensation. However, OPPS activity is only slightly increased
(threefold) by Triton. Also determined from these studies,
the bond-forming or bond-breaking step during IPP
condensation must be rate limiting because the first reaction
of C15 to C20, in which UPPS or OPPS is preincubated with
FPP so that the pyrophosphate dissociation and the
relocation of the intermediate are not involved in the
reaction, has the same rate as the subsequent steps [76].
Product distribution of cis-prenyltransferases
and trans-prenyltransferases
In the UPPS reaction, significant amounts of intermediates
are yielded as products under the conditions of 50 lM FPP
and 50 lM IPP, whereas the C55-UPP is synthesized with
5 lM FPP and 50 lM IPP. In contrast, OPPS produces no
intermediate products under these conditions [77]. Another
trans-prenyltransferase, SPPS from M. luteus, shows a
similar pattern of product distribution in which no intermediate is displaced from the active site at high concentration of FPP [15]. As OPPS and UPPS have similar FPP and
IPP Km values, OPPS and SPPS (trans-type) apparently
have higher affinities for intermediates compared with
UPPS (cis-type). At a fixed concentration of allylic substrate, the increased ratio of IPP to FPP results in the
synthesis of longer-chain products. As shown for a UPPS
from the Archaeon Sulfolobus acidocaldarius, the enzyme
mainly generates C50 and C55 when IPP is present in excess
of GGPP (or FPP), but decreasing the concentration of IPP
results in larger amounts of short-chain products [79]. This
could be due to the low affinity of IPP for the enzyme.
In the presence of Triton, UPPS mainly synthesizes C55
product. In contrast, under conditions in which product
release is slow, chain elongation beyond normal can occur.
Therefore, in the absence of Triton, UPPS could produce
C55–C75 polymers [76]. For the microsomal DDPPS of rat
liver, the chain length of products shifted downward from
C90 and C95 with increasing concentration of detergent [80].
The trans-type OPPS could also generate C40–C60 compounds as the final products. Derived from pre-steady-state
kinetic studies, the rate constant for condensation of an extra
IPP with C55 to produce C60 is fivefold lower than that for
regular IPP condensation (e.g. C50 to C55) in the UPPS
reaction (Fig. 6). On the other hand, the rate for elongation
from C40 to C45 catalyzed by OPPS is 100 times slower than
for elongation from C35 to C40 (Fig. 6). The trans enzyme
Fig. 6. Comparison of the kinetic pathways of UPPS and OPPS. OPPS
catalyzes elongation of C15 to C40, resulting in all trans double bonds,
and UPPS catalyzes formation of the C55 product containing newly
formed cis double bonds. The IPP condensation steps have rate constants of 2 s)1 and 2.5 s)1 for OPPS and UPPS, respectively. The
similar rate constants suggest closely related reaction mechanisms and
perhaps similar active-site environments. However, OPPS has higher
product specificity as judged from its much lower rate of conversion
into C45 extra product.
seems to have a more rigid active site than the cisprenyltransferase, as shown by the higher product specificity.
The most surprising observation on the product distribution of the UPPS and OPPS reactions is that, when the
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Structure, mechanism and function of prenyltransferases (Eur. J. Biochem. 269) 3347
concentration of the UPPS and FPP complex is in 10-fold
excess over that of IPP, long-chain polymers larger than C20
(GGPP), including C55, are generated. Pan et al. [76]
proposed that UPPS–intermediate complexes may have
greater affinity than the UPPS–FPP complex for the limited
amount of IPP, leading to the formation of product with the
correct chain length. OPPS shows the same phenomenon in
producing C20–C40 under the same conditions [77]. This
strategy for synthesizing the desired chain length seems to be
shared by both types of enzyme.
CLASS II: PROTEIN
PRENYL-TRANSFERASES
Structure and active site of protein prenyltransferases
Ras FTase is a Zn2+-dependent prenyltransferase containing a and b heterodimer, which catalyzes the farnesylation
on a C-terminal CaaX motif of the Ras protein. As shown
in Fig. 9B, the Zn2+-activated thiolate of Cys acts as a
nucelophile to attack the ionized farnesyl group. In the 3D
structure of a mammalian Ras FTase, both subunits are
largely composed of a helices (Fig. 7A) [37]. The a-2 to a-15
helices in the a subunit fold into a novel helical hairpin
structure, resulting in a crescent-shape domain that envelopes part of the b subunit. On the other hand, the 12 helices
of the b subunit form an a–a barrel. Six additional helices
connect the inner core of helices and form the outside of the
helical barrel. A deep cleft surrounded by hydrophobic
amino acids in the center of the barrel is proposed as
the FPP-binding pocket. A single Zn2+ ion is located at the
junction between the a-hydrophilic surface groove near the
subunit interface and the deep cleft in the b subunit. This
Zn2+ ion is pentaco-ordinated by the Asp297 and Cys299
located in the N-terminal helix 11, His362 in helix 13 of the
2 b subunit, and a water molecule as well as a bidentate ligand
Asp297b. Replacement of Cys299b with Ala results in lower
Zn2+ affinity and abolishes enzyme activity [81]. A nineamino-acid portion of the adjacent b subunit in the crystal
lattice was found to bind in the positively charged pocket of
the b subunit close to the FPP site. This observation allowed
the authors to speculate that the nonapeptide mimics some
aspects of normal CaaX peptide binding. This was supported by the fact that the first four residues of the peptide form
a type 1 b turn which indeed is similar to the observed
conformation for the natural CaaX peptide when bound to
FTase [82]. Furthermore, the C-terminal residue of the
nonapeptide could form hydrogen bonds with Lys164a,
Arg291b, and Lys294b.
Since the report of the first FTase structure, the same
research group has published the structure of FTase in
complex with FPP [38] as well as the structure of the ternary
complex containing an inactive FPP analogue and a CaaX
peptide substrate [39]. Many features of the apo-FTase
structure are retained in the complexes. According to the
structure of FTase in complex with FPP, the highly
conserved residues Trp303b, Try251b, Trp102b, Tyr205b,
and Tyr200a form hydrophobic interactions with FPP.
Arg202b in the binary complex adopts a different conformation from the structure of the apoenzyme to further
interact with the substrate. This residue is stabilized by
Asp200b and Met193b which also adopts a different
conformation. From the binary structure, two additional
amino acids, Cys254b and Gly250b, participate in the
binding of FPP. The diphosphate moiety of the FPP
substrate is hydrogen-bonded with His248b, Arg291b, and
Tyr300b. Lys164a and Lys294b are also within hydrogenbonding distance of the diphosphate. Mutations of His248b,
Arg291b, Lys294b, Tyr300b, and Trp303b cause 3–15 times
increased FPP Kd compared with the wild-type FTase [83],
consistent with the crystal structure. On the other hand,
replacement of Lys164a with Asn results in a markedly
decreased kcat value, suggesting that this residue plays a
catalytic role [84]. From the binary structure, Lys164 may
interact with the diphosphate moiety of FPP and be
involved in the transfer of Cys thiol to C1 of the substrate.
As for the binding of the 5th CaaX peptide substrate,
the structure of FTase complexed with a-hydroxyfarnesyl-
Fig. 7. (A) Ribbon diagram of Ras FTase and (B) the molecular ruler mechanism for substrate specificity of FTase and GGTase. (A) The heterodimeric
enzyme consists of two subunits, a and b, colored in cyan–blue and yellow–green, respectively. Most of the secondary structures are helices, with the
a subunit comprising seven helical hairpins that surround the more compact b subunit. The peptide substrate, colored red, binds to a cleft between
the two subunits, and so does the substrate analogue, colored magenta. The active site is located in the b subunit. It contains a zinc ion, shown in
blue, which is bound by three residues Asp297, Cys299 and His362, shown in cyan, and also makes bonds with the substrate molecules. (B) When
FPP is replaced with GGPP in the active site of FTase, the thiolate nucleophile is further away from the electrophilic carbon next to the
pyrophosphate leaving group, thereby decreasing the enzyme activity. Similarly, FPP is a poor substrate for GGTase.
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3348 P.-H. Liang et al. (Eur. J. Biochem. 269)
phosphonic acid and acetyl-Cys-Val-Ile-selenoMet-COOH
reveals that it binds in a cleft located in the subunit interface,
in agreement with the pervious apoenzyme structure [37].
The peptide is in contact with FPP and several amino acids
of the enzyme. The Ile-Met portion interacts with the
adjacent Tyr166a, and the carbonyl group of the main chain
forms a hydrogen bond with Arg202b. The Cys sulfur atom
of the peptide co-ordinates with a water molecule and the
Zn2+ ion which is stabilized by interacting with Asp297a,
Cys299b, and His362b. The N-terminal acetyl group makes
no contacts with the enzyme.
As revealed by the FTase structure in complex with
FPP, large amino-acid residues (Trp102b and Tyr205b)
are located on the bottom of the hydrophobic funnel for
FPP binding. The distance from these amino acids to the
bound Zn2+ ion is approximately the length of FPP from
C1 to C15 (Fig. 7B). According to this model, if a C20
GGPP is within the active site, its pyrophosphate is out of
the reach of Zn2+. This explains why GGPP binds
competitively with FPP to FTase but only FPP serves as
an effective substrate. On the other hand, FPP binds to
GGTase with 330-fold less affinity than GGPP [85].
However, it still serves as a moderately effective substrate
because its diphosphate moiety can be situated around the
Zn2+. This hypothesis has been confirmed by the solving
of the 3D structure of Rab GGTase [40]. The GGPP is
bound in the central cavity of the a–a barrel in the
b subunit with its diphosphate head group to the positively
charged cluster composed of Arg232b, Lys235b, and
L105a. The diphosphate is closed to the Zn2+, which is
co-ordinated with Asp238b, Cys240 b and His290b in a
similar way to that observed in FTase, and an additional
His residue. The structure of Rab GGTase can be
superimposed on that of Ras FTase. One of the most
striking differences is that on the bottom of the GGTase
active-site cavity, Ser48b and Leu99b replace the more
bulky Trp102b and Tyr154b seen in FTase, thereby
enlarging the active site to accommodate GGPP. This is
consistent the molecular ruler mechanism (Fig. 7B) proposed in FTase for substrate specificity.
Rab GGTase is unique and different from FTase and
type I GGTase in that it is able to prenylate Rab only in the
presence of Rab escort protein. It exclusively modifies
members of a single subfamily of Ras-related small GTPase,
the Rab proteins involved in the regulation of intracellular
vesicular transport in the biosynthetic secretory and exocytic/endocytic pathways [86,87]. Upon binding with the
protein complex, the Rab GGTase transfers two GGPPs to
the two Cys residues of Rab C-terminal -CC, -CXC, -CCX,
or -CCXX motif [88]. As shown in the superimposition of
the GGTase and FTase structures, several residues in the
peptide-binding pockets are different, reflecting their peptide substrate specificity.
Reaction mechanism and kinetics of protein:
prenyltransferase
Kinetic analysis of the enzymatic prenylation reaction
reveals a relatively fast chemical step 0.8–12 s)1 followed
by rate-limiting product release (0.06 s)1) [89,90]. Substrate
binding follows an ordered sequential mechanism with the
formation of the FTase–FPP complex before binding of the
CaaX substrate [91]. When the FPP analogue with a strong
electron-withdrawing fluorine atom replacing the C3
hydrogen was used, the reaction rate was significantly
decreased, suggesting formation of a carbocation at C1
during the reaction [92,93]. This phenomenon is similar to
that previously observed for FPPS as described above, but
the fluorinated FPP had less effect in the FTase reaction
than in the FPPS reaction. The formation of the carbocation with the simultaneous separation of the pyrophosphate of FPP represents in part the rate-determining step in
the FTase reaction as the metal required for co-ordination
with Cys of the peptide substrate is also involved in the
catalysis. The direct contact of Zn2+ with Cys thiol is
evident from the crystal structure of the FTase ternary
complex and the studies using the Co2+-substituted FTase
[94]. The use of the more thiophilic Cd2+ to increase the
thio affinity and lowering its pKa to display lower activity
suggests the direct participation of a metal ion in FTase
catalysis [95]. However, inclusion of high concentrations of
Mg2+ in the reaction mixture increases enzyme activity
700-fold because excess Mg2+ occupies a separate site,
facilitating departure of the diphosphate group [96].
Therefore, an associated character with partially positive
charge at C1 of FPP and partially negative charge
pyrophosphate oxygen as well as in the metal-co-ordinated
thiolate was proposed as the transition state of the FTase
reaction (see Fig. 9B).
CLASS III: TERPENOID CYCLASES
General structure and mechanism of terpenoid cyclase
Terpenoid cyclases such as squalene cyclase, pentalenene
synthase, 5-epi-aristolochene synthase, and trichodiene
synthase are responsible for the synthesis of cholesterol, a
hydrocarbon precursor of the pentalenolactone family of
antibiotics, a precursor of the antifungal phytoalexin
capsidiol, and the precursor of antibiotics and mycotoxins,
respectively. The last three enzymes catalyze the cyclization
of FPP involving: (a) ionization of FPP to an allylic cation
which acts as electrophile to react with one of the p bonds of
the substrate for cyclization; (b) relocalization of the
carbocation via hydride transfer and Wagner-Meerwein
rearrangements; (c) deprotonation or capture of an exogenous nucleophile such as water to eliminate the carbocation. On the other hand, squalene synthase catalyzes the
cyclization of squalene, which is formed by coupling two
FPP molecules. Like trans-type IPPSs, which make linear
polymers from FPP, the four cyclases also contain conserved Asp-rich motifs, suggesting that these enzymes have
similar strategies for activating FPP. In the structures of
these three enzymes, the similar structural feature referred to
as Ôterpenoid synthase foldÕ with 10–12 mostly antiparallel
a helices is found, as also observed in IPPS and FTase
(Fig. 8). The high structural similarity provides support for
the hypothesis that the three families of prenyltransferases
described in this review have related evolution despite their
low sequence similarity.
Pentalenene synthase
As shown in Fig. 8A, the active site of bacterial pentalenene
synthase cloned from Streptomyces UC5319 [97] is located
in a hydrophobic pocket with the bottom sealed by aromatic
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Structure, mechanism and function of prenyltransferases (Eur. J. Biochem. 269) 3349
Fig. 8. Ribbon representation of the structures of cyclic terpenoid-synthetic enzymes pentalenene synthase (A), 5-epi-aristolochene synthase (B),
trichodiene synthase (C) and squalene synthase (D). They are shown in different colors with the N-terminus and C-terminus labeled. All of these
enzymes have the terpenoid synthase fold and contain a helices as their major secondary-structure elements. The homologous helices that constitute
the active site are emphasized with broad ribbons.
residues Phe57, Phe76, and Trp308, and aliphatic residues
Leu53, Val177, Val179, Thr182, and Val301 [43]. These
residues together create the necessary conformation for FPP
to undergo cyclization to pentalenene. The top of the pocket
is surrounded by hydrophilic amino acids, including His309,
Asn219, Arg44, Arg157, Arg173, Lys226, Arg230, and
particularly Asp80 and Asp84 with their carboxylate
protruded into the active site to complex with Mg2+.
Together they facilitate the FPP carbocation formation as
shown in Fig. 9C (path a). Subsequent cyclization and
carbocation rearrangement processes use a His309 catalytic
residue in the active site [43].
5-epi-Aristolochene synthase
Tobacco 5-epi-aristolochene synthase adopts entirely an
a-helical structure with short loops and turns (Fig. 8B). The
structure folds into two domains: the N-terminal domain
aligns structurally with two glycosyl hydroxylases, and its
function is not known; the C-terminal domain shares the
common fold with FPPS. The active site of epi-aristolochene
synthase is located in the C-terminal domains containing two
Mg2+-binding sites. The Asp301 and Asp305 found in the
conserved DDxxD motif co-ordinate with a Mg2+ [42]. The
Asp444, Thr448, Glu452 and a water molecule co-ordinate
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3350 P.-H. Liang et al. (Eur. J. Biochem. 269)
interaction with Arg264, Arg441 and the three Mg2+ ions.
The newly formed allylic carbocation is positioned near the
main-chain carbonyl oxygens of residues 401 and 402 at
the kink in helix G, and the C1 is near the p orbitals of the
C10–C11 bond. The bond forming between C1 and C10
leaves a tertiary carbocation on C11 (see Fig. 9C, path b),
which is stabilized by Tyr527. A deprotonation at C13 by
Asp525 leads to the germacrene intermediate. The formation of a C–C bond between C2 and C7 of the intermediate
is triggered by protonation of C6 with a proton donated by
Tyr520. This leaves a positive charge on C3 to form a
bicyclic eudesmane carbocation intermediate. The following
rearrangement of the carbocation is facilitated by the
electron-rich side chain of Trp273 to generate the final
product aristolochene.
Trichodiene synthase
Fig. 9. The transition state for reactions of cis-IPPS and trans-IPPS
(A), protein farnesyltransferase (B), and pentalenene synthase, epi-aristolochene synthase, and trichodiene synthase (C). In three cases, the
carbocation intermediate at C1–C3 carbons of FPP is formed, and the
negative charge developed on pyrophosphate is stabilized by co-ordination with Mg2+. The deprotonated IPP or a Zn2+-activated thiolate
anion of a protein acts as a nucleophile to attack the carbocation at C1
resulting in condensation with departure of the pyrophosphate. In the
cyclase reaction, the nearby C¼C bond donates electrons for ring
closure. The paths a, b, and c denote the routes of electron migration
from the C¼C bond to C1 with the release of pyrophosphate catalyzed
by pentalenene synthase, epi-aristolochene synthase, and trichodiene
synthase, respectively.
with the other Mg2+. These two Mg2+ ions located in the
entrance of the active-site pocket are responsible for binding
to the FPP substrate. A number of hydrophobic amino acids
around the FPP site were proposed to provide further
hydrophobic binding interactions.
The catalytic mechanism of epi-aristolochene synthase
was elucidated by its structure in complex with the substrate
analogue farnesyl hydroxyphosphonate. Accordingly, as a
loop (residues 521–534) becomes ordered, it forms a lid that
clamps down over the active-site entrance in the presence of
farnesyl hydroxyphosphonate. This brings the loop containing Arg264 and Arg266 close to the C1 hydroxy group
of farnesyl hydroxyphosphonate, and an additional Mg2+
site forms. In analogy, the released negatively charged
diphosphate group of the FPP substrate is stabilized by
The recently solved 3D structure of trichodiene synthase
(Fig. 8C) reveals 17 a helices, six of which (C, D, G, H, I,
and J) form the hydrophobic active-site cleft [44]. An Asprich D100DSKD motif, which is important for catalytic
activity as suggested by the mutagenesis studies [98], is
located in the active site at the C-terminus of helix D.
Three Mg2+ ions were found in the electron map of the
enzyme–pyrophosphate complex. Among them, two metal
ions in the active-site region co-ordinate with Asp100 (the
first residue of the conserved Asp-rich motif). The
conserved Asp residues are also found in aristolochene
synthase and FPPS. The other Mg2+ is located in the site
of N225, S229, and E233 on helix H, which are in a
consensus sequence conserved among all known terpene
synthase sequences. The metal ions are bound to the
enzyme in the presence of pyrophosphate, and the
pyrophosphate (also probably FPP) triggers a protein
conformational change, in which D101 breaks a salt link
with R62 and forms a new salt bridge with R304 after
pyrophosphate binding. The purpose of the protein
conformational change is proposed to facilitate the departure of the pyrophosphate group of the substrate. These
ligand-induced conformational changes were also observed
in other terpene synthases such as 5-epi-aristolochene
synthase, suggestive of divergent evolution from a common
ancestor. On the basis of the structures and the mechanism
of trichodiene synthase, a model of the enzyme reaction
has been constructed (Fig. 9C). Path c is the initial step of
the reaction; the recapture of a-phosphate oxygen by the
C3 cation forms the intermediate, which then undergoes
dephosphorylation and C1–C6 bond formation [44]. The
resulting C7 carbocation attracts the electrons of the
C10–C11 p orbital to form the C7–C11 bond. Subsequently, three carbocation-driven migrations occur to
generate the final product.
Squalene cyclase
Squalene cyclase and 2,3-oxidosqualene synthase catalyze
the cyclization reactions to give steroid compounds and
have become the targets for development of anticholesteremic and antifungal drugs. Bacterial squalene synthase
had been crystallized in three distinct forms, and the
structure of form A solved. The enzyme is a homodimer,
and the structure shows two domains in the a subunit
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A large number of isoprenoids have significant biological
functions in nature. The structural and mechanistic information available so far provides an initial step to understanding the functions of many other prenyltransferases and
a basis for designing enzyme agonists and antagonists to
treat various diseases. However, the structures and even
functions of many isoprenoid-synthetic enzymes, as well as
the proteins catalyzing and undergoing prenylation, are still
not known. Further studies on the structure, mechanism
and function of prenyltransferases are therefore required.
(Fig. 8D) [41]. Domain 1 is an a6–a6 barrel of two
concentric rings of parallel a helices, which is similar to the
fold of FPPSs and glucanases [99]. Domain 2 forms an a–a
barrel and inserts into domain 1. The active site is located
in a central cavity, with two amino acids in a DxDD motif,
Asp376 and Asp377, on the top of the cavity [41].
Mutagenesis of these two Asp residues abolishes enzyme
activity, indicating their importance in catalysis [100].
Based on the structure, Asp376 is the catalytic acid to
protonate the first C¼C double bond, resulting in a
carbocation for the initiation of ring cyclization. A water
molecule acts as a base to receive a proton on the other
end of the squalene molecule, which is polarized by the
other water in a hydrogen-bonding network formed by
Glu45 and Gln262. A constriction formed by Phe166,
Val174, Phe434, and Cys435 in a mobile loop may serve as
a gate for substrate passage.
CONCLUSION
This review summarizes the 3D structures, IPP condensation kinetics and mechanism of trans-IPPS and cis-IPPS for
the production of linear polyprenyl pyrophosphates, and
the reaction mechanism and structures of protein prenyltransferases and isoprenyl diphosphate cyclases. As illustrated in Fig. 9, the three families of prenyltransferases have
similar strategies for substrate binding and catalysis. First,
they use hydrophobic interactions to bind the hydrocarbon
moiety of the allylic pyrophosphate substrate. Secondly,
pyrophosphtae leaving is facilitated by co-ordination of
active-site amino acids such as Asp, Glu and Lys with
Mg2+. Although the protein FTase was isolated with Zn2+
bound, the inclusion of Mg2+ greatly enhances enzyme
activity. The carbocation character of the allylic substrate
(FPP) exists during reactions of all these prenyltransferases.
In the IPPS reaction, IPP with a proton abstracted attacks
the carbocation of FPP. For the protein farnesyltransferase,
a thiolate anion of the substrate protein or a peptide acts as
the nucelophile. The FPP cyclase reaction is also initiated by
the formation of an FPP carbocation which attracts a pair
of electrons from the nearby C¼C double bond for the
ring closure (paths a, b, and c for pentalenene synthase,
5-epi-aristolochene synthase, and trichodiene synthase,
respectively).
As the X-ray structure of cis-type UPPS has become
available, the general mechanism for product chain-length
determination of the cis and trans forms of IPPS has
gradually been understood. As revealed by the 3D
structures, the size of the active-site cavity apparently
controls the chain length of the final product of the IPPS
reaction. Several large amino acids form a floor to seal the
bottom of the active site and block further elongation of
the product. Site-directed substitution of key large amino
acids with smaller ones extends the size of the active site,
thereby allowing the formation of a longer product and
vice versa. The substrate specificities of protein FTase and
GGTase are controlled by the size of the substrates. The
larger GGPP is more distant from the Zn2+ catalytic site
when binding on FTase and hence produces lower activity.
The accurately tailored active sites of FPP cyclases
(pentalenene synthase, 5-epi-aristolochene synthase, and
trichodiene synthase) guarantee formation of the correct
cyclization products.
ACKNOWLEDGEMENTS
We are grateful to Yi-Kai Chen for the preparation of the sequence
alignment figures.
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