Báo cáo khoa học: An alternative mechanism of product chain-length determination in type III geranylgeranyl diphosphate synthase docx

An alternative mechanism of product chain-length determination
in type III geranylgeranyl diphosphate synthase
Hisashi Hemmi, Motoyoshi Noike, Toru Nakayama and Tokuzo Nishino
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Miyagi, Japan
(All-E) prenyl diphosphate synthases catalyze the con-
secutive condensation of isopentenyl diphosphates with
allylic prenyl diphosphates, producing products with vari-
ous chain-lengths that are unique for each enzyme. Some
short-chain (all-E) prenyl diphosphate synthases, i.e.
farnesyl diphosphate synthases and geranylgeranyl
diphosphate synthases contain characteristic amino acid
sequences around the allylic substrate binding sites, which
have been shown to play a role in determining the chain-
length of the product. However, among these enzymes,
which are classified into several types based on the pos-
sessive patterns of such characteristics, type III geranyl-
geranyl diphosphate synthases, which consist of enzymes
from eukaryotes (excepting plants), lack these features.
In this study, we report that mutagenesis at the second
position before the conserved G(Q/E) motif, which is
distant from the well-studied region, affects the chain-
length of the product for a type III geranylgeranyl
diphosphate synthase from Saccharomyces cerevisiae.This
clearly suggests that a novel mechanism is operative in the
product determination for this type of enzyme. We also
show herein that mutagenesis at the corresponding posi-
tion of an archaeal medium-chain enzyme also alters its
product specificity. These results provide valuable infor-
mation on the molecular evolution of (all-E)prenyl
diphosphate synthases.
Keywords: prenyltransferase; geranylgeranyl diphosphate

synthase; hexaprenyl diphosphate synthase; mutagenesis;
molecular evolution.
(All-E) prenyl diphosphate synthases catalyze consecutive
condensations of isopentenyl diphosphate (IPP) in the
E-type configuration with allylic primer substrates and yield
products with various hydrocarbon-chain lengths that are
specific to each enzyme. The products are utilized as
precursors for numerous types of isoprenoid compounds
such as steroids, carotenoids, respiratory quinones and
prenylated proteins (Fig. 1). The enzymes have been
classified into three groups based on their quaternary
structure and the chain-length of the product produced, i.e.
short-, medium-, and long-chain enzymes yielding C
10)25
,
C
30)35
and C
40)50
products, respectively [1,2]; therefore,
these designations are used in this paper. The enzymes of the
three groups are thought to have similar structures and the
same catalytic mechanism is involved, because their amino
acid sequences have a high degree of similarity [3,4]. For
example, two aspartate-rich motifs, designated as FARM
(the first aspartate-rich motif) and SARM (the second
aspartate-rich motif), are completely conserved among these
enzymes and act as binding sites for allylic substrates and
IPP, respectively. A crystallographic analysis of avian
farnesyl diphosphate synthase (FPS) revealed that the

homodimeric enzyme consists almost entirely of a-helices,
some of which constitute a reaction cavity in the center of a
subunit of the enzyme, and that FARM and SARM both
exist on distinct a-helices and face each other at different
sides of the rim of the cavity [5].
Short-chain (all-E) prenyl diphosphate synthases are
known to have strict product specificities, and the mecha-
nisms involved in product determination have so far been
investigated using FPSs and geranylgeranyl diphosphate
synthases (GGPSs) that yield C
15
and C
20
products,
respectively. Several mutagenic studies have revealed that
aromatic amino acids, frequently found at the fourth and
fifth positions, before FARM of short-chain enzymes, are
involved in these mechanisms [6–12]. It is thought that these
aromatic amino acids act as the bottom of a reaction cavity
to prevent further elongation of the prenyl chain of the final
product. In addition, two amino acids inserted into FARM,
which occurs in FPSs and GGPSs from bacteria and plants,
are also considered to be involved in the mechanism [9].
Thus, we designated the area, including FARM and several
amino acids upstream of it, as the CLD (chain-length
determination) region and proposed a classification of these
enzymes based on the patterns of such characteristic amino
acid residues (Fig. 2). However, no such residue is found in
enzymes classified as type III GGPSs, the group consisting
of GGPSs from eukaryotes (excepting plants). The typical

Correspondence to Tokuzo Nishino, Department of Biomolecular
Engineering, Graduate School of Engineering, Tohoku University,
Aoba-yama 07, Sendai, Miyagi 980-8579, Japan.
Fax/Tel.: + 81 22 217 7270,
E-mail:
Abbreviations: IPP, isopentenyl diphosphate; GPP, geranyl
diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl
diphosphate; FPS, farnesyl diphosphate synthase; GGPS,
geranylgeranyl diphosphate synthase; HPS, hexaprenyl diphosphate
synthase; FARM, the first aspartate-rich motif; CLD, chain-length
determination.
Enzymes: geranylgeranyl diphosphate synthase (EC 2.5.1.29),
hexaprenyl diphosphate synthase (EC 2.5.1.30), farnesyl diphosphate
synthase (EC 2.5.1.10).
(Received 24 January 2003, revised 10 March 2003,
accepted 18 March 2003)
Eur. J. Biochem. 270, 2186–2194 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03583.x
sequence of the CLD region of type III GGPSs resembles
that of medium- and long-chain (all-E) prenyl diphosphate
synthases: no bulky aromatic amino acids exist at the fourth
and fifth positions before FARM, and the insertion of two
amino acids is not observed. This fact strongly suggests that
some characteristic amino acids that are located outside the
CLD region are conserved in type III GGPSs and play an
important role in the mechanism of chain-length determin-
ationintheseenzymes.
In our previous report in 1996, which was the first
reference to the mechanism of product determination in
(all-E) prenyl diphosphate synthase, mutants of type II FPS
from Bacillus stearothermophilus were produced by random

mutagenesis, and those yielding longer products such as
geranylgeranyl diphosphate (GGPP) were selected [6]. Two
of four selected mutants shared the same amino acid
substitution at the fifth position before FARM (Y81H),
which led us to subsequently discover the importance of the
Fig. 1. Reactions of (all-E) prenyl diphosphate synthases in isoprenoid biosynthesis. OPP, OP
2
O
5
3–
.
Fig. 2. Alignment of amino acid sequences around FARM and the G(Q/E) motif of various (all-E) prenyl diphosphate synthases. The partial amino
acid sequences of the enzymes classified into two types of FPSs, three types of GGPSs and longer-chain enzymes are aligned. Sce FPS, S. cerevisiae
FPS; Gga FPS, Gallus gallus (avian) FPS; Eco FPS, E. coli FPS; Bst FPS, B. stearothermophilus FPS; Sac GGPS, S. acidocaldarius GGPS; Mth
GGPS, Methanobacterium thermoautotrophicum GGPS; Pan GGPS, Pantoea ananatis (Erwinia uredovora) GGPS; Ath GGPS, Arabidopsis thaliana
GGPS; SceGGPS, S. cerevisiae GGPS; Hsa GGPS, Homo sapiens GGPS; SsoHPS, S. solfataricus HPS (hexaprenyl diphosphate synthase); Eco
OPS, E. coli octaprenyl diphosphate (C
40
) synthase. The amino acid residues that were suggested to concern product determination are shaded.
Arrowheads indicate the positions into which substitutive mutations were introduced in this work.
Ó FEBS 2003 Another evolutionary route of prenyltransferase (Eur. J. Biochem. 270) 2187
CLD region in product determination. However, the rest of
the mutants had substitutive mutations at different posi-
tions. One mutant, designated as mutant 3, possessed
mutations at two positions (V157A/H182Y), and further
mutational experiments revealed that a V157A mutation
was sufficient to change product specificity. The mutated
site is located at the second position before the G(Q/E)
motif that is highly conserved among all (all-E)prenyl
diphosphate synthases. This fact led us to the hypothesis

that the position might play an important role in product
determination in type III GGPSs because they do not
possess the usual characteristic amino acid residues in the
CLD region. This idea is supported by the fact that
histidine, a relatively large amino acid, is conserved at this
position in type III GGPSs, while smaller ones, such as
valine, alanine, serine, cysteine or glutamic acid, are
conserved in longer-chain enzymes and in the short-chain
enzymes of the other groups (Fig. 2).
In this study, substitutive mutations were introduced at
the second position before the G(Q/E) motif of a type III
GGPS from Saccharomyces cerevisiae. The mutations were
clearly shown to affect the product determination of the
enzyme. Mutational studies using an archaeal medium-
chain enzyme, hexaprenyl diphosphate synthase (HPS)
from Sulfolobus solfataricus, also provided support for the
importance of this specific position and, moreover, suggest
the existence of a novel evolutional pathway of (all-E)
prenyl diphosphate synthases.
Materials and methods
Materials
Precoated reversed-phase thin-layer chromatography plates,
LKC-18F were purchased from Whatman Chemical
Separation, Inc. (All-E)-GGPP (all-E)-farnesyl diphosphate
(FPP), and geranyl diphosphate (GPP) were kindly donated
by K. Ogura and T. Koyama, Tohoku University.
[1-
14
C]IPP was purchased from Amersham Bioscience Inc.
All other chemicals were of analytical grade.

General procedures
Restriction enzyme digestions, transformations, and other
standard molecular biological techniques were carried out
as described by Sambrook et al.[13].
Site-directed mutagenesis
A DNA fragment including the S. cerevisiae GGPS gene
was amplified by means of the polymerase chain reaction
utilizing the S. cerevisiae genome as a template, KOD DNA
polymerase (Toyobo), and the primers indicated below:
ScGGPS-Fw, 5¢-TAGACGGTACCAAGCTT
CATATG
GAGGCCAAGATAGATGAGC-3¢;ScGGPS-Rv,5¢-GT
CTAGGTACCAAGCTT
GGATCCTCACAATTCGGA
TAAGTGGTC-3¢. The sequences corresponding to the
NdeIandBamHI sites used later are underlined. The
amplified fragment was cleaved with these two restriction
enzymes and then inserted into the NdeI/BamHI-treated
pET-15b vector (Novagen) to construct pET-HisScGG, a
plasmid for the recombinant expression of His
6
-tagged
S. cerevisiae GGPS. On the other hand, pET-HisHPS,
constructed in our previous study [14], was employed in the
recombinant expression of S. solfataricus HPS.
Substitutive mutations were specifically introduced into
these plasmids utilizing a QuickChange Mutagenesis Kit
(Stratagene) according to the protocol provided by the
manufacturer. Pairs of oligonucleotide primers used for
each mutagenesis are indicated in Table 1. In addition,

plasmids for the expression of mutants possessing additional
mutations with mutant H139A, i.e. pET-HisScGG-H139A-
3A, pET-HisScGG-H139A-4A and pET-HisScGG-
H139A-3A4A, were constructed from the plasmid
pET-HisScGG-H139A.
Expression and purification of the wild-type
and mutated enzymes
Escherichia coli BL21(DE3) transformed with each of the
plasmids shown above or in Table 1 was cultivated in
50 mL of M9YG broth supplemented with ampicillin
(50 mgÆL
)1
). When the D
600
of the culture reached 0.5, the
transformed bacteria were induced with 1.0 m
M
isopropyl
thio-b-
D
-galactoside. After an additional overnight cultiva-
tion, the cells were harvested. The disruption of the cells and
the purification of the tagged enzymes were conducted
utilizing a MagExtractor His-tag Kit (Toyobo) according to
the protocol recommended by the manufacturer. The level
of purification was determined by 15% SDS/PAGE (data
not shown).
Measurement of prenyltransferase activity
The assay mixture for wild-type and mutant S. cerevisiae
GGPSs contained, in a final volume of 200 lL, 0.5 nmol

[1–
14
C]IPP (2 GBqÆmmol
)1
), 0.5 nmol of an allylic primer
(GGPP, FPP or GPP), 1 lmol MgCl
2
,2lmol Tris/HCl
buffer (pH 7.5), 2 lmol KF, and a suitable amount of each
enzyme. This mixture was incubated at 30 °C for 1.5 h, and
the reaction was stopped by chilling the mixture in an ice
bath. The mixture was shaken with 600 lL of 1-butanol
saturated with H
2
O. The butanol layer was washed with
water saturated with NaCl, and the radioactivity in 100 lL
of the butanol layer was measured with a TRI-CARB 1600
liquid scintilation counter (Packard). The residual butanol
layer was used for product analysis.
S. solfataricus HPS activity was assayed using the same
method, except that the reaction mixture for the wild-type
and mutants of S. solfataricus HPS contained 0.5 nmol
[1–
14
C]IPP (2 GBqÆmmol
)1
), 0.5 or 25 nmol GPP, 1 lmol
MgCl
2
,2lmol of a phosphate buffer (pH 6.3), 0.1% (v/v)

Triton X-100, and a suitable amount of enzyme in a final
volume of 200 lL. It was incubated at 55 °C for 30 min and
then processed as described above.
Product analysis
Prenyl diphosphates in the residual 1-butanol layer were
treated with acid phosphatase according to the method of
Fujii et al. [15]. The hydrolysates were extracted with
pentane and analyzed by reversed-phase thin-layer chro-
matography using a precoated plate, LKC-18F, developed
with acetone/H
2
O (9 : 1, v/v). Authentic, standard alcohols
2188 H. Hemmi et al.(Eur. J. Biochem. 270) Ó FEBS 2003
were visualized with iodine vapor, and the distribution
of radioactivity was detected by Molecular imager
(Bio-Rad).
Results
Mutational studies using
S. cerevisiae
GGPS
To confirm the hypothesis that the amino acid at the second
position before the G(Q/E) motif plays an important role in
the product determination of type III GGPSs, a system for
the recombinant expression of S. cerevisiae GGPS, which is
classified as a type III GGPS, was constructed. Mutational
experiments were conducted using the recombinant
enzymes. The wild-type and mutant enzymes expressed
by E. coli were purified utilizing a (His)
6
-tag fused at the

N-terminus of each enzyme. We initially constructed
mutants of S. cerevisiae GGPSinwhichhistidine139,
which is located at the second position before the G(Q/E)
motif, was replaced with smaller amino acids such as
alanine, glycine or serine (Fig. 3A). These mutants retained
high enzyme activities that are comparable with that of wild-
type GGPS when GPP or FPP was used as the allylic
substrate (Fig. 3B). One of the mutants, H139A, showed
considerable activity when GGPP was used, while the wild-
type GGPS barely reacted with the allylic substrate.
Analyses demonstrated that the H139A mutant yielded a
longer C
25
final product (Fig. 3C). Moreover, the mutant
produced a small amount of C
30
prenyl diphosphate when
GGPP was used as the allylic substrate. The H139G and
H139S mutants also yielded C
25
prenyl diphosphate, but in
very small amounts. This strongly suggests that the
mechanism involved in the product determination in
S. cerevisiae GGPS is largely dependent on histidine 139,
a residue that is completely different from those which play
major roles in the mechanisms of many short-chain
enzymes.
Based on the above data, we hypothesize that S. cerevis-
iae GGPS utilizes a mechanism that is similar to those of
many short-chain enzymes, in which bulky amino acids

before FARM block the elongation of the prenyl chain at
the bottom of the cavity in the enzyme. Thus, additional
mutations were introduced into mutant H139A to deter-
mine whether the cavity of the enzyme can be expanded
directly below position 139. It has been reported that
Sulfolobus acidocaldarius GGPS mutants that possess
double mutations, i.e. the replacement of the amino acids
at the fifth and eighth positions before FARM with smaller
ones, are not able to control the chain-length of their final
products and yield products longer than C
100
[10]. Similar
mutational experiments using B. stearothermophilus FPS
provided the same result. These mutated positions are
thought to exist at the same side of the a-helix that contains
FARM, which binds the diphosphate moiety of prenyl
diphosphate. The introduction of smaller amino acids at
these positions would be expected to give rise to a path
through which the prenyl chain could elongate along the
a-helix. According to the three-dimensional structure of
avian FPS (Protein Data Bank accession no. 1FPS), which
is the only (all-E) prenyl diphosphate synthase whose crystal
structure has been determined, the a-helix containing the
G(Q/E) motif is in the same orientation as that containing
FARM. These two a-helices adjoin each other and comprise
a portion of the reaction cavity. Therefore, we replaced
L135 and/or I136 of mutant H139A with alanine because
the distance between H139 and these residues generally
corresponds to a pitch of an a-helical coil (Fig. 4A). All
of the mutants L135A/H139A, I136A/H139A and

Table 1. Mutagenic primers used in this work.
Primers Sequences
a
Generated plasmids/mutants
For the construction of S. cerevisiae GGPS mutants
ScGGPS-H139A-Fw 5¢-GAATTGATCAATCTAgc
ccgcGGACAAGGCTTGG-3¢ pET-HisScGG-H139A/
ScGGPS-H139A-Rv 5¢-CCAAGCCTTGT
CCgcgggcTAGATTGATCAATTC-3¢ mutant H139A
ScGGPS-H139G-Fw 5¢-GAATTGATCAATCTAgg
ccgcGGACAAGGCTTGG-3¢ pET-HisScGG-H139G/
ScGGPS-H139G-Rv 5¢-CCAAGCCTTGT
CCgcggccTAGATTGATCAATTC-3¢ mutant H139G
ScGGPS-H139S-Fw 5¢-GAATTGATCAATCTAag
ccgcGGACAAGGCTTGG-3¢ pET-HisScGG-H139S/
ScGGPS-H139S-Rv 5¢-CCAAGCCTTGTCCgcggctTAGATTGATCAATTC-3¢ mutant H139S
ScGGPS-H139A-3A-Fw 5¢-CGATTTTCAAC
GAgGAgTTGgcCAATCTAGCCCGCGG-3¢ pET-HisScGG-H139A-3A/
ScGGPS-H139A-3A-Rv 5¢-CCGCGGGCTAGATTGgcCAA
cTCcTCGTTGAAAATCG-3¢ mutant I136A/H139A
ScGGPS-H139A-4A-Fw 5¢-CGATTTTCAAC
GAgGAggcGATCAATCTAGCCCG-3¢ pET-HisScGG-H139A-4A/
ScGGPS-H139A-4A-Rv 5¢-CGGGCTAGATTGATCgc
cTCcTCGTTGAAAATCG-3¢ mutant L135A/H139A
ScGGPS-H139A-3A4A-Fw 5¢-CGATTTTCAAC
GAgGAggcGgcCAATCTAGCCCGCGG-3¢ pET-HisScGG-H139A-3A4A/
ScGGPS-H139A-3A4A-Rv 5¢-CCGCGGGCTAGATTGgcCgc
cTCcTCGTTGAAAATCG-3¢ mutant L135A/I136A/H139A
For the construction of S. solfataricus HPS mutants
SsHPS-S140H-Fw 5¢-GTTATGGAAAGACACCcatGTGG

GaGCTCTAAGGGATATG-3¢ pET-HisHPS-S140H/
SsHPS-S140H-Rv 5¢-CATATCCCTTAGAGCtCCCACatgGGTGTCTTTCCATAAC-3¢ mutant S140H
SsHPS-S140V-Fw 5¢-GTTATGGAAAGACACCgtAGTGG
GaGCTCTAAGGGATATG-3¢ pET-HisHPS-S140V/
SsHPS-S140V-Rv 5¢-CATATCCCTTA
GAGCtCCCACTacGGTGTCTTTCCATAAC-3¢ mutant S140V
SsHPS-S140F-Fw 5¢-GTTATGGAAAGACACCTttGTGG
GaGCTCTAAGGGATATG-3¢ pET-HisHPS-S140F/
SsHPS-S140F-Rv 5¢-CATATCCCTTA
GAGCtCCCACaaAGGTGTCTTTCCATAAC-3¢ mutant S140F
SsHPS-S140Y-Fw 5¢-GTTATGGAAAGACACCTatGTGG
GaGCTCTAAGGGATATG-3¢ pET-HisHPS-S140Y/
SsHPS-S140Y-Rv 5¢-CATATCCCTTAGAGCtCCCACatAGGTGTCTTTCCATAAC-3¢ mutant S140Y
a
Mismatched sequences are lower-cased, and newly introduced restriction sites to confirm mutagenesis are underlined.
Ó FEBS 2003 Another evolutionary route of prenyltransferase (Eur. J. Biochem. 270) 2189
L135A/I136A/H139A yielded longer products than H139A
when GGPP was used as the allylic substrate (Fig. 4B). The
longest products of these mutants appeared to be C
40
or
more. This result strongly suggests that the prenyl chain of
the product elongates along the a-helix containing the
G(Q/E) motif in the mutants and that residue H139 acts as
the bottom of the reaction cavity in the wild-type S. cere-
visiae GGPS.
Fig. 3. Replacement of the amino acid residue at the second position before the G(Q/E) motif in S. cerevisiae GGPS. (A) Partial amino acid sequences
around the G(Q/E) motif of wild type and mutated enzymes are aligned. The substituted amino acid residues are shaded. (B) Specific activities of the
wild-type and mutated enzymes. Enzyme reactions were carried as described in Materials and methods, and activities were determined by the
amount of radioactivity extracted with 1-butanol. (C) TLC autoradiochromatograms of the reaction products of the wild-type and mutated

enzymes. The products were analyzed as described in Materials and methods. The allylic substrate used was indicated at the top of each
autoradiochromatogram. Lane 1, mutant H139A; lane 2, mutant H139G; lane 3, mutant H139S; lane 4, wild type GGPS. Under all assay
conditions, less than 30% of each substrate reacted. Ori., origin; S.F., solvent front.
2190 H. Hemmi et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Mutational studies using
S. solfataricus
HPS
In our previous phylogenetic study, S. solfataricus HPS, a
medium-chain enzyme that was recently cloned and char-
acterized, was shown to exist at an anomalous position in
the phylogenetic tree of (all-E) prenyl diphosphate synthases
[16]. The enzyme was included in a branch consisting of
eukaryotic short-chain enzymes, i.e. type I FPSs and type III
GGPSs, but not in that of other medium- and long-chain
enzymes. Its phylogenetic position was particularly close to
enzymes classified as type III GGPSs, which strongly
suggests a close relationship between the archaeal medium-
chain enzyme and type III GGPSs in the evolution of (all-E)
prenyl diphosphate synthases. However, the issue of what
difference between their amino acid sequences gave rise to
the difference in their final products remains unclear. It must
be noted that our attempt to change the product specificity
of the medium-chain enzyme by replacing the CLD region
with that of S. cereviseae GGPSfailed[14].Asaresult,
S. solfataricus HPS was used for mutational analyses to
demonstrate the importance of the amino acid residue
corresponding to H139 of S. cerevisiae GGPS and to
confirm the evolutionary relationship between the archaeal
HPS and the eukaryotic GGPS.
S. solfataricus HPS contains a serine residue at position

140, the second position before the G(Q/E) motif (GA in
S. solfataricus HPS represents an exception); thus, S140 was
replaced with a larger amino acid to determine whether this
substitutive mutation could be the origin of the change in
product specificity of the medium-chain enzyme to those of
short-chain enzymes. We constructed two mutants, S140H
and S140V, that mimic S. cerevisiae GGPS and B. stearo-
thermophilus FPS, respectively, because the product speci-
ficity of both of these short-chain enzymes changes as the
result of a point mutation at their corresponding positions
(Fig. 5A). In addition, we also constructed two mutants,
S140F and S140Y, containing an aromatic amino acid
residue at this position. When the same concentrations of
IPP and GPP were used in the assays, the enzyme activity of
the S140H and S140Y mutants was decreased to 54 and
37% of that of the wild-type HPS, respectively, while the
other mutants showed an activity comparable with that of
the wild-type enzyme. Product analyses showed that their
final products continued to be the hexaprenyl diphosphate,
the C
30
product (Fig. 5B). However, except for S140V, the
mutants accumulated large amounts of FPP as an inter-
mediate. On the other hand, the S140V mutant showed a
slightly increased production of GGPP, in comparison with
the wild-type enzyme. When the concentration of GPP was
increased to 50 times that of IPP to enhance the production
of intermediates, all of the mutants showed an enzyme
activity comparable to the wild-type enzyme. However, all
mutants produced FPP as the main product, while the wild-

type HPS yielded the C
30
product (Fig. 5C). Only the S140V
mutant continued to produce a considerable amount of
hexaprenyl diphosphate. These results indicate that position
140 plays a significant role in the product determination in
these mutants, probably by blocking the elongation of the
prenyl chain of the products, but in an incomplete manner.
We hypothesize that type III GGPSs might have evolved
from some longer-chain enzyme, such as S. solfataricus
HPS, based on the result of a phylogenetic analysis of (all-E)
prenyl diphosphate synthases. The fact that the product
specificity of the archaeal medium-chain enzyme partially
changed into those of short-chain enzymes by the acquisi-
tion of a characteristic amino acid residue at the second
position before the G(Q/E) motif supports this hypothesis.
Discussion
The importance of the second position before the G(Q/E)
motif in the mechanism of product determination in
S. cerevisiae GGPS was examined by mutagenic studies.
The results from the characterization of the mutants
strongly suggests that the amino acid residue at the position
of S. cerevisiae GGPS might function in a manner similar to
those of the aromatic amino acids at the fourth and fifth
positions before FARM in some other short-chain enzymes,
Fig. 4. Introduction of additional mutations into mutant H139A. (A)
Partial amino acid sequences around the G(Q/E) motif of mutant
H139A and mutated enzymes are aligned. The substituted amino acid
residues are shaded. (B) TLC autoradiochromatograms of the reaction
products of the wild-type and mutated enzymes. The products were

analyzed as described in Materials and methods using GGPP as the
allylic substrate. Lane 1, mutant H139A; lane 2, mutant I136A/
H139A; lane 3, mutant L135A/H139A; lane 4, mutant L135A/I136A/
H139A; lane 5, wild type GGPS. Under all assay conditions, less than
30% of each substrate reacted. Ori., origin; S.F., solvent front.
Ó FEBS 2003 Another evolutionary route of prenyltransferase (Eur. J. Biochem. 270) 2191
i.e. the formation of the bottom of a reaction cavity (Fig. 6).
However, this hypothesis appears to be incompatible with
the fact that the H139G mutant did not yield products
longer than those produced by H139A although the
substitution with the smallest glycine residue would be
expected to have the largest effect on the expansion of the
cavity. On the other hand, the size of the side-chain of the
amino acid residue used to substitute for serine 140 of
S. solfataricus HPS seemed to correlate negatively with the
product chain-length. This is similar to results obtained
from an experiment in which the fifth position before
FARM of B. stearothermophilus FPS was replaced [8]. The
inconsistent result observed with the mutants of S. cerevis-
iae GGPS might arise from a structural change in the cavity
of the H139G mutant, which could arise from the
introduction of a flexible glycine residue.
The second position before the G(Q/E) motif would be
expected to play a major role in the mechanism of product
determination in type III GGPSs because these enzymes do
not contain characteristic amino acids such as aromatic
residues in the CLD region. It is known that a relatively
large amino acid residue, i.e. histidine, is conserved at the
position of the thus-far-characterized type III GGPSs, while
smaller amino acid residues such as valine, alanine, serine,

cysteine or glutamic acid are conserved in the longer-chain
(all-E) prenyl diphosphate synthases and in the short-chain
enzymes of other groups. This fact strongly suggests that all
type III GGPSs share the same mechanism of product
determination, in which the histidine residue is involved.
However, previous studies have proposed that the second
position before the G(Q/E) motif might also be involved in
product determination in short-chain enzymes other than
type III GGPSs. For example, we previously reported that a
point mutation at this position in B. stearothermophilus
FPS, which is classified as a type II FPS, results in the
elongation of the final product [6]. Kawasaki et al. recently
reported that the product specificities for FPS from
Streptomyces argenteolus (DNA Data Bank of Japan
accession no. AB083108) and GGPS from Streptomyces
greseolosporeus (AB037907), both of which anomalously
contain CLD regions similar to those found in type I
GGPSs, are interchangeable by simply exchanging their
amino acids at the second position before the G(Q/E) motif
[17]. In the three-dimensional structure of avian FPS, E182,
the residue at the second position before the G(Q/E) motif,
is in spatial proximity to F112 and F113, the residues at the
fourth and fifth positions before FARM, respectively. The
side chain of F112 is in direct contact with that of E182. This
suggests that, in some short-chain enzymes, there might be
Fig. 5. Introduction of substitutive mutations into the second position
before the G(Q/E) motif in S. solfataricus HPS. (A) Partial amino acid
sequences around the G(Q/E) motif of wild type and mutated enzymes
are aligned. The substituted amino acid residues are shaded. The
reaction products of the enzymes were analyzed as described in

Materials and methods using 0.5 nmol (B) and 25 nmol (C) of GPP as
the allylic substrate. Lane 1, mutant S140H; lane 2, mutant S140V;
lane 3, mutant S140F; lane 4, mutant S140 Y; lane 5, wild type HPS.
Under all assay conditions, less than 40% of each substrate reacted.
Ori., origin; S.F., solvent front.
2192 H. Hemmi et al.(Eur. J. Biochem. 270) Ó FEBS 2003
some cooperation among these residues, as they relate to
product determination. It should be noted that Stanley
Fernandez et al. attempted to alter the product specificity of
avian FPS into that of geranyl diphosphate synthase by
introducing point mutations at position 181, the third
position before the G(Q/E) motif, although the mutations
resulted in a significant reduction in enzyme activity [18].
Moreover, as indicated herein, the replacement of serine at
the second position before the G(Q/E) motif with valine to
mimic type II GGPSs also affected the product specificity of
S. solfataricus HPS. Our future interest is to reveal the
importance of this position in the product determination
mechanisms of short-chain enzymes other than type III
GGPSs.
The mutational experiment utilizing S. solfataricus HPS
successfully demonstrated that the medium-chain enzyme
could be altered to yield short-chain products as the result of
a point mutation at this position. The S140H mutant
constructed in this experiment and type III GGPSs share
similar features at regions that are known to be involved in
product determination. Neither contains characteristic
amino acid residues in their CLD regions, such as bulky
amino acids at the fourth or fifth position before FARM or
the insertion of two amino acids into FARM, and both

contain a histidine residue at the second position before the
G(Q/E) motif. Based on the information obtained from the
phylogenetic tree of (all-E) prenyl diphosphate synthases, in
which S. solfataricus HPS shows a particularly close
relationship with type III GGPSs, we conclude that type
III GGPSs might have evolved from an ancestor resembling
S. solfataricus HPS [16]. It is conceivable that the introduc-
tion of the S140H mutation into S. solfataricus HPS
simulates the evolution of type III GGPSs from the
ancestral enzyme. At least, the change in product specificity
as the result of a mutagenesis at the second position before
the G(Q/E) motif represents a possible pathway of mole-
cular evolution between type III GGPSs and the longer-chain
enzymes, and the evolutionary pathway would be totally
independent of known ones that have arisen from mutations
in the CLD regions (Fig. 6).
Acknowledgements
This work was supported by Grants-in-Aid from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan. We are
grateful to Drs Kyozo Ogura and Tanetoshi Koyama, Tohoku
University, for providing prenyl diphosphates. We thank Dr Tohru
Dairi, Toyama Prefectural University, for his participation in helpful
discussions.
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