Download Functional characterization of LePGT1, a membrane

Biochem. J. (2009) 421, 231–241 (Printed in Great Britain)
231
doi:10.1042/BJ20081968
Functional characterization of LePGT1, a membrane-bound
prenyltransferase involved in the geranylation of p -hydroxybenzoic acid
Kazuaki OHARA*1 , Ayumu MUROYA†, Nobuhiro FUKUSHIMA† and Kazufumi YAZAKI*2
*Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Uji 611-0011, Japan, and †Science & Technology Systems, Inc., 1-20-1
Shibuya, Tokyo 150-0002, Japan
The AS-PT (aromatic substrate prenyltransferase) family plays a
critical role in the biosynthesis of important quinone compounds
such as ubiquinone and plastoquinone, although biochemical
characterizations of AS-PTs have rarely been carried out
because most members are membrane-bound enzymes with
multiple transmembrane α-helices. PPTs [PHB (p-hydroxybenzoic acid) prenyltransferases] are a large subfamily of AS-PTs
involved in ubiquinone and naphthoquinone biosynthesis.
LePGT1 [Lithospermum erythrorhizon PHB geranyltransferase]
is the regulatory enzyme for the biosynthesis of shikonin, a
naphthoquinone pigment, and was utilized in the present study as a
representative of membrane-type AS-PTs to clarify the function
of this enzyme family at the molecular level. Site-directed
mutagenesis of LePGT1 with a yeast expression system indicated
three out of six conserved aspartate residues to be critical to
the enzymatic activity. A detailed kinetic analysis of mutant
enzymes revealed the amino acid residues responsible for
substrate binding were also identified. Contrary to ubiquinone
biosynthetic PPTs, such as UBIA in Escherichia coli which
accepts many prenyl substrates of different chain lengths, LePGT1
can utilize only geranyl diphosphate as its prenyl substrate. Thus
the substrate specificity was analysed using chimeric enzymes
derived from LePGT1 and UBIA. In vitro and in vivo analyses
of the chimeras suggested that the determinant region for this
specificity was within 130 amino acids of the N-terminal. A 3D
(three-dimensional) molecular model of the substrate-binding site
consistent with these biochemical findings was generated.
INTRODUCTION
structure of a representative enzyme, NphB (Orf2), was revealed
[21,22], although there are no plant homologues of this soluble
type AS-PT. In contrast, little detailed molecular characterization
of membrane-bound AS-PTs has been reported regardless of
origin. In particular, no characterization of plant AS-PTs has been
reported. This is mainly because of the difficulty in handling these
proteins.
Representative of membrane-bound AS-PTs are the PPT [PHB
(p-hydroxybenzoic acid) prenyltransferase] group. Genes coding
for these enzymes have been identified in many organisms from
prokaryotes to eukaryotes, and are mostly responsible for UQ
biosynthesis [23–28]. The reaction catalysed by PPT is proposed
to be a rate-limiting step in UQ biosynthesis [29], and all
members characterized thus far show a broad specificity for prenyl
diphosphates, in accepting substrates of different chain lengths.
The only exception known to date is LePGT1 (Lithospermum
erythrorhizon PHB geranyltransferase) [30], which only accepts
GPP (geranyl diphosphate) and is involved in the biosynthesis
of a red naphthoquinone, shikonin, a plant secondary metabolite.
LePGT1 was reported to be the key regulatory enzyme of shikonin
biosynthesis [31] and is not relevant to the formation of UQ [30].
The present study utilized LePGT1 to characterize the
enzymatic function of membrane-bound AS-PTs by site-directed
mutagenesis, as it has several advantages over other membranetype AS-PTs. For example, the expression of the recombinant
protein in yeast was established, with which the enzymatic activity
can be analysed quantitatively with a wide dynamic range using
GPP as the native prenyl substrate, and it is an ER (endoplasmic
The prenyltransferase family contains a large number of
membrane-intrinsic proteins, which represent enzymes that accept
various aromatic substrates. These AS-PTs (aromatic substrate
prenyltransferases) catalyse the substitution of an aromatic
proton with a prenyl group, leading to the formation of benzoand naphtho-quinones as well as prenylated polyphenols, which
play various important biological roles in organisms ranging
from bacteria to humans. For example, AS-PTs catalyse the key
reaction in the biosynthesis of naturally occurring quinines, such
as UQ (ubiquinone) [1], plastoquinone [2,3], and also vitamin E
[4,5], in which their basic skeletons are formed via prenylated
aromatic intermediates. In addition, prenylation reactions contribute to the diversification of plant aromatic natural products in
terms of chemical structure and biological activity. For example,
flavonoids [6–11], coumarins [12,13], and polyketides [14] are
frequently prenylated, and cannabinoid [15] also represents a
biologically active prenylated aromatic in higher plants.
Some prenylated flavonoids are involved in plant defence
mechanisms as phytoalexins [16,17], and some are beneficial
to human health, exhibiting anti-cancer, anti-bacterial and antityrosinase activities etc., leading to their potential use as natural
medicines [18–20]. Due to these attractive properties of prenylated
compounds and the crucial role of prenyl moieties in their
biological activities, intensive studies have been carried out.
In fact, some soluble-type AS-PT genes involved in antibiotic
biosynthesis have been cloned from Streptomyces and the crystal
Key words: aromatic substrate prenyltransferase, coenzyme Q,
mutagenesis, shikonin, substrate specificity, ubiquinone.
Abbreviations used: 3D, three-dimensional; AS-PT, aromatic substrate prenyltransferase; FARM, first aspartate-rich motif; FPP, farnesyl diphosphate;
GPP, geranyl diphosphate; LC, liquid chromatography; LePGT1, Lithospermum erythrorhizon PHB geranyltransferase; PHB, p -hydroxybenzoic acid; PPT,
PHB prenyltransferase; SARM, second aspartate-rich motif; UQ, ubiquinone.
1
Present address: Central Laboratories for Frontier Technology, Kirin Holdings Co., Ltd., 1-13-5 Fukuura Kanazawa-ku, Yokohama-shi, Kanagawa
236-0004, Japan.
2
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2009 Biochemical Society
232
Table 1
K. Ohara and others
The oligonucleotide sequences are listed below, where underlining indicates non-native restriction sites for subcloning
Oligonucleotide
Sequence
PGT1_EcoRI_Fw
PGT1-myc_XhoI_Rv
UBIA_EcoRI_Fw
UBIA-myc_XhoI_Rv
LePGT1_PstI_Fw
LePGT1_MfeI_Rv
UBIA_XbaI_Fw
UBIA_XbaI_Rv
UBIA_SacI_Fw
UBIA_SacI_Rv
5 -cgcgaattcAGAAATGGTTTCCAGCAAACAAAC-3
5 -cgctcgagCTACAGATCCTCTTCTGAGATGAGTTTTTGTTCAGGAAACAATCTTCCAAGTAAG-3
5 -cgcgaattcAGAAATGGAGTGGAGTCTGACGCAGAATA-3
5 -cgctcgagCTACAGATCCTCTTCTGAGATGAGTTTTTGTTCGAAATGCCAGTAACTCATTGCCAG-3
5 -cgctgcagATGGTTTCCAGCAAACAAACACAG-3
5 -cgcaattgGCTTGTCTAACCTTGCTAGATGAGC-3
5 - ggtctagaCCACTTCCCAGCGGCGCG -3
5 - ggtctagaCGCCGTGCGCTTAACATG-3
5 - gcgagctCCAATGGCTTTTGCCGCTG-3
5 -gcgagctccCCAGCCAAACGCCGCGCCC-3
reticulum)-localized protein without a signal peptide, which can
cause trouble in yeast [30]. To clarify the molecular basis of
the difference in prenyl-substrate specificity, several chimeric
enzymes were also prepared using GPP-specific LePGT1 and
a UQ biosynthetic PPT, UBIA with broad substrate specificity
[23,32], and enzymatic function was characterized using an
in vitro enzyme assay and in vivo complementation test with
a yeast mutant. Moreover, a 3D (three-dimensional) molecular
model of the substrate-binding domain of LePGT1 was generated,
which is consistent with the biochemical data obtained.
EXPERIMENTAL
Construction of expression vectors for yeast
A pDR196 vector [33] was used for the expression of LePGT1
in yeast, also for point mutants of LePGT1, UBIA, and chimeric
enzymes. All recombinant enzymes were expressed as fusion proteins with a c-Myc tag at the C-terminus for immunodetection.
Addition of the nucleotide sequence encoding the c-Myc
epitope was carried out by PCR using the following primers,
PGT1_EcoRI_Fw, PGT1-Myc_XhoI_Rv, UBIA_EcoRI_Fw and
UBIA-Myc_XhoI_Rv, and the sequences are listed in Table 1. The
PCR products were subcloned into pDR196 via the EcoRI and
XhoI restriction sites. Site-directed mutagenesis was performed
using the QuikChange site-directed mutagenesis kit (Stratagene)
according to the manufacturer’s instructions. The PCR template
for the mutagenesis was pDR196-LePGT1, and the introduction of
the mutations was confirmed by nucleotide sequencing of the
entire LePGT1 gene. The mutagenic oligonucleotides designed to
produce the desired point mutations are listed in Supplementary
Figure S1 (at http://www.BiochemJ.org/bj/421/bj4210231add.
htm).
Chimeric enzymes derived from LePGT1 and UBIA were
created by combinations of restriction digestion and PCR. The
parental LePGT1 and UBIA genes subcloned in pDR196 were
digested with EcoRI/SacI, EcoRI/XbaI, XbaI/SacI or PstI/MfeI.
DNA fragments of the correct sizes were purified from an agarose
gel and ligated with the appropriate DNA fragments that were
prepared by PCR to create chimeric cDNAs. To amplify the DNA
fragments of LePGT1 or UBIA for ligation, the following primers
were used, LePGT1_PstI_Fw, LePGT1_MfeI_Rv, UBIA_
XbaI_Fw, UBIA_XbaI_Rv, UBIA_SacI_Fw, UBIA_SacI_Rv,
PGT1_EcoRI_Fw, PGT1-Myc_XhoI_Rv, UBIA_EcoRI_Fw and
UBIA-Myc_XhoI_Rv (Table 1). All chimeric cDNAs were
sequenced completely to confirm the absence of sequence errors.
copy of the COQ2 gene (strain coq2 [30]) to eliminate
the background AS-PT activity. The microsomal preparation
from S. cerevisiae, and measurements of PPT activity using
non-radioactive or radioisotope-labelled substrates were made
according to Yazaki et al. [30] with some modifications. The
standard reaction mixture used to determine levels of enzymatic
activity was 50 mM Tris/HCl (pH 7.6) containing 400 μM GPP,
400 μM PHB and 10 mM MgCl2 in a total volume of 200 μl.
After incubation at 30 ◦C for 60 min, the reaction was terminated
by the addition of 5 μl of formic acid. The reaction product was
extracted with 150 μl of ethyl acetate containing testosterone
propionate as an internal standard. This organic phase was
evaporated dry, dissolved in 50 μl of methanol and analysed
by HPLC as described previously [30]. Lineweaver–Burk plots
were employed to calculate the K m from the HPLC data. With
a radioactive substrate of [14 C]PHB, the reaction products were
analysed by silica gel TLC (Kiesel gel, Merck, 20 cm × 20 cm)
with a solvent system of toluene/ethyl acetate (4:1). The TLC
plates were exposed to an imaging plate (Fuji Film) at room
temperature (22 ◦C) for 5 days and then analysed with a BAS1800
image analyser (Fuji Film).
Detection of UQ6 from S. cerevisiae
UQ6 produced in yeast cells was extracted according to the
method of Uchida et al. [34] with slight modifications. LC (liquid
chromatography)-MS was carried out on a Shimadzu model
2010A system liquid chromatograph and mass spectrometer
equipped with a atmospheric-pressure chemical ionization source,
in which an LC-10AD solvent delivery system was used as the
LC unit under the following conditions: column LiChrosphere
100RP-18 (Merck) 4 × 250 mm; solvent system, ethanol/2propanol (1:1); flow rate, 0.2 ml/min. UQ6 was identified by direct
comparison with a standard specimen.
Immunoblotting
A monoclonal antibody against the Myc epitope was purchased
from Cell Signaling Technology (Danvers, MA, U.S.A.).
SDS/PAGE and immunoblotting of microsomal membrane
proteins was performed according to a method reported previously
[35] with slight modifications. Aliquots (20 μg) of yeast microsomal fractions were used for SDS/PAGE. Protein concentrations were determined according to the method of Bradford [36],
using BSA as the standard.
Measurements of PPT activity
3D structural modelling of the catalytic centre of LePGT1
All point mutants and chimeric enzymes were constitutively
expressed in Saccharomyces cerevisiae containing a disrupted
A sequence alignment between FPP (farnesyl diphosphate)
synthase and LePGT1 was prepared based on similarity around
c The Authors Journal compilation c 2009 Biochemical Society
Mutational analyses of membrane-bound prenyltransferase
233
Figure 1 Representative features of LePGT1 protein and a multiple alignment of regions conserved among PPT family members involved in UQ and shikonin
biosynthesis
(A) Putative transmembrane α-helices (TM) and positions of Regions I–III. TM domains are indicated in black and numbered from the N-terminus. (B) Amino acid residues corresponding to Regions
I II, and III are shown. The multiple alignment was made using ClustalW (http://clustalw.ddbj.nig.ac.jp/top-j.html). Asterisks indicate the residues mutated in this study. Double asterisks are aspartate
residues exclusively conserved in the Mg2+ -dependent prenyltransferase family. At, Arabidopsis thaliana ; Ce, Caenorhabditis elegans ; Hs, Homo sapiens ; Mm, Mus musculus ; Os, Oryza sativa ; Sp,
Streptococcus pneumoniae . (C) Immunoblotting of microsomal proteins that were used in this study. To detect the recombinant protein, all point mutants as well as wild-type LePGT1 were expressed
as fusion proteins with a c-Myc epitope tag at the C-terminus, and a Myc-tag monoclonal antibody (Cell Signaling Technology) was used for immunoblotting.
the FARM (first aspartate-rich motif) and the SARM (second
aspartate-rich motif). The crystal structure of FPP synthase (PDB
ID: 2F8Z) was used for molecular modelling. A tentative model
of LePGT1 was built using the DS MODELLER module on
a Discovery studio 1.7 (Accelrys). The docking simulation of
magnesium ions in the catalytic centre of the LePGT1 model
was performed with CDOCKER. The three magnesium ions with
the top three scores are represented in Figure 8. The docked
structure showed two magnesium ions that bound to the Oδ atoms
of Asp84 , Asp87 and Asp91 , and one magnesium ion bound to
the Oδ atoms of Asp208 and Asp212 . The model was optimized
by 500 steps of potential energy minimization with CHARMm
force field. PHB and GPP were docked into the model of LePGT1
including the magnesium ions. These models of ternary complex
were optimized by 500 steps of potential energy minimization
with CHARMm force field. Models were drawn using PyMOL.
RESULTS
Site-directed mutagenesis of LePGT1 (alanine-scan)
Comparative topological analyses with five different prediction
programs suggested that LePGT1 possesses nine transmembrane
α-helices (see Supplementary Figure S2 at http://www.BiochemJ.
org/bj/421/bj4210231add.htm). LePGT1 possesses three highly
conserved amino acid sequences designated Regions I, II and III
in the hydrophilic loops of the representative topology model
predicted by the TMHMM program (http://www.cbs.dtu.dk/
services/TMHMM/), which are shown in Figure 1 [26,30,37].
c The Authors Journal compilation c 2009 Biochemical Society
234
K. Ohara and others
Figure 2 PHB geranyltransferase activity of LePGT1 mutants in which
conserved amino acids were substituted with alanine (alanine scan)
The PHB geranyltransferase activities of fourteen mutant enzymes were measured using the
microsomal fraction from S. cerevisiae transformants. The y -axis indicates specific activity
(nmol/h per mg of protein), with relative activity (%) compared to the activity of wild-type
LePGT1 (100 %) also shown. ud, undetectable.
All these conserved hydrophilic loops face one side of the
biological membrane, suggesting that the conserved domains
interact to mediate enzyme function, such as substrate recognition.
In order to investigate the role of each conserved amino acid, sitedirected mutations were introduced into LePGT1. Fifteen highly
conserved and three non-conserved amino acid residues were individually changed to the aliphatic amino acid alanine: Region I,
R76A, N83A, D84A, F86A, D87A, D91A and R96A; Region
II, K152A and R153A; Region III, D201A, Y204A, H206A,
Q207A, D208A, D211A, D212A, S219A and K229A (indicated
with asterisks in Figure 1B). These mutant enzymes as well
as the wild-type LePGT1 were heterologously expressed in S.
cerevisiae strain coq2, in which the endogenous PPT gene was
disrupted. The microsomal fraction was prepared first to check the
expression level of the recombinant proteins by immunoblotting
with an anti-Myc antibody to detect the c-Myc epitope tag
attached to the C-terminus of LePGT1. The results showed that
13 mutant proteins had almost the same level of accumulation as
the wild-type enzyme, whereas D91A accumulated less than the
wild-type enzyme. Recombinant proteins were not detectable for
four mutants (F86A, D201A, H206A and Y204A) (Figure 1C),
which were not subjected to subsequent experiments.
The enzymatic activity of 14 mutants was measured using
GPP and PHB, the native substrates of LePGT1. The results are
summarized in Figure 2. All mutant enzymes showed less activity
than the wild-type protein, although the R153A mutant maintained
a high level of activity (81.6 %). The molecular modelling of
a bacterial orthologue, UBIA, revealed that this arginine residue
(Arg137 in UBIA) is important for the recognition of phosphate
groups via Mg2+ ions [37], but our biochemical data did not
support this hypothesis. The mutant enzymes, in which one of
four highly conserved aspartates was changed to alanine, did not
show detectable activity (D87A, D91A and D208A), or exhibited
extremely weak activity (D212A), i.e. 1000-fold lower than the
control. In addition, seven mutant enzymes, in which a conserved
amino acid was changed to alanine, showed only 0.15 % to
2.31 % of the level of activity of the wild-type, whereas S219A
and K229A showed appreciable activity, i.e. 14.7 % and 12.9 %,
respectively.
Next, we determined apparent K m values to investigate the affinity of mutant enzymes for GPP and PHB (Figure 3). The K m of
c The Authors Journal compilation c 2009 Biochemical Society
Figure 3 Comparison of K m values of LePGT1 point mutants by alanine
scanning
(A) K m values for PHB of LePGT1 alanine mutants were determined from Lineweaver–Burk
plots. (B) Those for GPP in LePGT1 alanine mutants are shown. Relative K m values compared
with wild-type LePGT1 are shown as percentages. n.d., not determined.
wild-type LePGT1 with the c-Myc tag was 66.4 μM for PHB and
29.5 μM for GPP. Among the mutant enzymes, N83A showed a
strong increase in the K m value for PHB (1426 μM), i.e. 21.5-fold
that of the wild-type, although its K m value for GPP (38.3 μM)
was almost the same (1.3-fold) as that of the wild-type. Another
mutant, R76A, also showed a strong decrease in affinity for PHB
(K m 268 μM, 4-fold higher than the control), whereas this mutant
revealed a 4-fold increase in affinity for GPP. In contrast, the K m
values for GPP were increased in other mutants, such as S219A
(147 μM, 5-fold) and K229A (88.6 μM, 3-fold); however, the
influence on the K m value for GPP appeared to be weaker than
that for PHB as compared with N83A (21.5-fold). The strong
influence of N83A of the affinity for PHB may suggest the crucial
involvement of Asn83 in the recognition of the PHB molecule. In
fact, this asparagine residue is conserved in prenyltransferases
accepting PHB as a substrate, but not in the homogentisate
prenyltransferase family. The present study could not determine
the K m value for PHB in R96A or the K m values for PHB and GPP
in D212A because of the weak activities of these mutant enzymes.
Substitution of four exclusively conserved aspartates with similar
amino acids
To investigate the role of the conserved aspartate residues (Asp87 ,
Asp91 , Asp208 and Asp212 ) in more detail, these residues were
replaced with a similar amino acid: glutamate (namely D87E,
D91E, D208E and D212E) or asparagine (namely, D87N, D91N,
D208N and D212N). These eight mutant enzymes were expressed
Mutational analyses of membrane-bound prenyltransferase
235
Figure 4 Substitution of four conserved aspartate residues with glutamate
or asparagine
Activities of mutated enzymes in which one of four exclusively conserved aspartate residues
(Asp87 , Asp91 , Asp208 and Asp212 ) was replaced with glutamate (E) or asparagine (N) as similar
amino acids. For comparison, the activity of the alanine (A) mutants is also shown. In addition
to specific activity (nmol/h per mg of protein), relative activity compared with wild-type LePGT1
is also shown as a percentage. K m values of wild-type and mutated LePGT1 for PHB and GPP
are indicated above each column. ud, undetectable; n.e., not expressed.
in S. cerevisiae coq2, and their expression at the protein level
was assessed by immunoblotting using the microsomal fraction
(Figure 1C). The mutated proteins D208E, D208N and D212E
were detected at the same level as the wild-type, whereas D87E
had a lower level than the wild-type. However, no recombinant
protein was detected for D87N, D91E, D91N or D212N.
The activity of mutant proteins was confirmed as shown in
Figure 4. No detectable geranyltransferase activity was found
in D87E or D208N, although these recombinant proteins were
clearly detected by immunoblotting. In contrast, D208E and
D212E showed enzymatic activity, although only 0.41 %
and 3.05 % respectively of that of the wild-type. These findings
indicate the importance of the conserved aspartate residues to vary,
i.e. the substitution with glutamate at Asp208 and Asp212 sustained
the catalytic function, whereas the substitution at Asp87 abolished the activity. The K m values of D208E and D212E showed that
the affinity for both substrates was the same as (D212E for GPP)
or higher than that of the wild-type (D212E for PHB; D208E for
both) (Figure 4).
Substitution of other highly conserved residues with similar amino
acids
In addition to the four exclusively conserved aspartate residues,
the alanine scan experiments indicated that other highly conserved
amino acids could also be important for the catalytic mechanisms
(Figure 2). To investigate the enzymatic function of other
conserved amino acids, in the present study we tried to prepare
six mutant enzymes, namely N83D, D84E, D84Q, R96K, K152R
and K229R. The reason why these amino acids were selected was
as follows: Asn83 had a strong influence on the K m value for PHB,
Asp84 was used for comparison with the four conserved aspartate
residues, Arg96 and Lys152 strongly affected the enzymatic activity
as shown in Figure 2, and Lys229 is arginine in the mouse
orthologue and the necessity of the lysine residue was to be
confirmed. After the monitoring of protein levels using the c-Myc
tag, N83D, D84E, D84Q and R96K were found to be successfully
expressed in the host strain S. cerevisiae coq2, whereas
the K152R and K229R mutants failed to accumulate in the
recombinant yeast (Figure 1C).
Subsequently, enzymatic activity was measured and the
apparent K m values for GPP and PHB were determined with
Figure 5 Substitution of conserved but non-essential residues with a
similar amino acid
Activities of mutated enzymes in which one of the conserved residues: (A), Asn83 ; (B), Asp84 ;
(C), Arg96 was substituted with a similar amino acid. For comparison, the corresponding alanine
mutants are also shown. The y -axis is specific activity (nmol/h per mg of protein). K m values of
wild-type and mutated LePGT1 for PHB and GPP are indicated.
these four mutant enzymes, which were compared with the
alanine-substituted mutants (Figure 5). N83D showed a 7.4fold higher level of activity than N83A, whereas its affinity for
PHB (K m = 70.0 μM) was the same as that of the wild-type,
although N83A lost affinity for PHB, i.e. 21-fold lower than the
D-substituted mutant (Figure 5A). The substitution of Asp84 with
glutamate was much more preferable, namely 12-fold greater
geranyltransferase activity than D84A; in contrast, D84Q showed
a strong decrease in activity (Figure 5B). This finding suggests
that an acidic amino acid at position 84 is crucial to maintain
a high level of enzymatic activity. However, the K m values of
D84E for the two substrates (PHB, 230 μM; GPP, 207 μM) were
approx. 3.5- and 6.5-fold higher than those of the wild-type, and
also apparently higher than those of D84A. The size of the amino
acid would also be important for recognition of the substrate.
The substitution of arginine at position 96 significantly reduced
the enzymatic function. Even the change to a similar basic
amino acid, lysine, brought about a substantial loss of activity,
demonstrating that this arginine residue is important not only in
providing a positive charge at this position but also in the size
of the side chain. It would be interesting to compare the K m
values for the substitutions at Arg96 , particularly with regard to
the bulk of the side chain, but unfortunately reliable quantitative
data were not obtained due to the detection limit of the reaction
product.
Analyses of substrate specificity with chimeric enzymes derived
from LePGT1 and UBIA
The main difference between LePGT1 and other PPTs involved
in UQ biosynthesis is the availability of prenyl diphosphates
c The Authors Journal compilation c 2009 Biochemical Society
236
Figure 6
K. Ohara and others
PHB geranyltransferase activity and K m values of chimeric enzymes derived from LePGT1 and UBIA
(A) Amino acid sequences of LePGT1 and UBIA. Arrowheads indicate the junctions for chimeras of LePGT1 and UBIA. Conserved Regions I, II, and III are underlined. (B) Schematic drawing of
chimeric enzymes and PHB geranyltransferase (GT) activities. ud, undetectable. (C) K m values of chimeric enzymes for PHB and GPP.
with various chain lengths, namely only LePGT1 is strictly GPPspecific, and all other characterized members of this family show
broad specificity for prenyl substrates. However, the substrate
preference was not influenced by a single amino acid substitution
created in the above experiments (results not shown). Thus, to
clarify the regions responsible for the difference in substrate
specificity in the primary structure, several chimeric enzymes
were prepared using LePGT1 and UBIA, the PPT of UQ
biosynthesis in Escherichia coli [23] (Figures 6A and 6B). The
native prenyl substrate of UBIA is octaprenyl diphosphate (C40),
but UBIA is also able to utilize GPP as a prenyl substrate due
to its broad specificity [32]. Utilizing measurements of PHB
c The Authors Journal compilation c 2009 Biochemical Society
geranyltransferase activity as a standard, the chimeric enzymes
were expressed and their activity was detected. However, the
activity could only be evaluated for Chimera 1 and Chimera 4
(Figure 6B), because the functional expression of these chimeras
in yeast was very limited, i.e. some were not expressed at the
protein level, and some showed no enzymatic activity. With
chimeric enzymes 1 and 4, the K m values for both substrates
were compared to those of the wild-type enzymes, UBIA and
LePGT1. UBIA and Chimera 4 showed a considerably higher K m
for GPP (3080 μM and 990 μM) than LePGT1 and Chimera 1
(29.5 μM and 33.5 μM), whereas these four enzymes had similar
K m values for PHB (Figure 6C).
Mutational analyses of membrane-bound prenyltransferase
Figure 7
237
Specificity of chimeric enzymes for prenyl substrates and in vivo complementation testing of coq2
(A) PHB farnesyltransferase activities of LePGT1, UBIA and chimeric enzymes. PHB farnesyltransferase activity relative to PHB geranyltransferase activity is shown as a percentage. (B) Each chimeric
enzyme, as well as wild-type LePGT1 and UBIA, was expressed in coq2, which was grown on an SD (Sabaraud dextrose) plate with either glucose (Glu) or glycerol (Gly) as the sole carbon source.
pDR196, empty vector; W303-1A, wild-type S. cerevisiae ; coq2, COQ2 gene disruptant of S. cerevisiae . (C) UQ6 detection of UQ6 in coq2-expressing chimeric enzyme. The molecular mass of
UQ6 (m /z 592) was scanned by LC-MS. Arrows indicate the retention time of UQ6.
To assess the prenyl substrate specificity, PHB farnesyltransferase activity was employed. The results showed FPP was not
a preferred substrate for Chimera 1 or for LePGT1 (2.20 % and
3.13 % of the activity of GPP); however, Chimera 4 as well as
UBIA could clearly utilize FPP as a prenyl substrate (105 % and
49.6 % of the activity of GPP) (Figure 7A). A yeast complement
test was also carried out. S. cerevisiae coq2 is defective in
producing UQ due to a lack of PPT, the key prenyltransferase
for the biosynthesis of the intermediate m-prenyl-p-hydroxyPHB, and this strain shows a clear growth defect on a minimal
medium containing glycerol as the sole carbon source, because
UQ is required to utilize this non-fermentable carbon source for
growth [24]. The expression of UBIA and Chimera 4 successfully
complemented the growth defect of the COQ2 disruptant on
glycerol-containing medium in nearly the same manner as in the
wild-type yeast, whereas no growth was observed for the coq2
strain harbouring either pDR-LePGT1 Chimera 1, or the empty
vector pDR196 (Figure 7B). As this bioassay is much more
sensitive than the HPLC assay, it was tested whether or not other
chimeras (Chimeras 2, 3 and 5–7) with no detectable activity
in vitro could compliment the growth defect on glycerol medium.
However, they also failed to rescue the growth of the coq2
strain on the glycerol plate (Figure 7B). In addition to the growth
complementation, the in vivo production of native UQ in yeast
was confirmed, i.e. UQ6, in the extract of yeast transformants
expressing Chimera 4 as well as UBIA used as a positive control
(Figure 7C). The analysis showed that UBIA and Chimera 4 could
indeed function as PHB hexaprenyltransferase in vivo leading to
the accumulation of UQ6 together with the growth recovery of
coq2 yeast on the glycerol plate.
DISCUSSION
Because LePGT1 is bound to the membrane via nine putative
transmembrane α-helices, a structural analysis by X-ray
c The Authors Journal compilation c 2009 Biochemical Society
238
Figure 8
K. Ohara and others
3D structure of LePGT1 determined by molecular modelling
(A) Substrate-binding site of LePGT1. The polypeptide chain of LePGT1 is shown in yellow, the
geranyl moiety of GPP is in magenta, diphosphate is shown in orange, PHB is drawn in green,
and Mg2+ ions are shown as grey spheres. Amino acids affecting the catalytic function are also
indicated. The distance between phosphate and the OH residue of PHB, and the carbon atom at
the m -position of PHB and the C-1 atom of GPP are 2.76 Å and 7.11 Å respectively. The Figure
was drawn using PyMOL. (B) Surface model of LePGT1. The geranyl chain of GPP is shown in
magenta, and the N-terminal region (amino acids 41–170), which is involved in the substrate
specificity for GPP, is drawn in cyan.
crystallography was inapplicable. Thus a molecular modelling
approach was attempted to visualize its 3D structure or at
least that of the catalytic domain including bound substrates.
Among various Mg-dependent prenyltransferases, FPP synthase
provided a suitable model for generating a reasonable 3D structure
of LePGT1 because this polypeptide consisted of more than
10 α-helices despite being a soluble protein. More importantly,
FPP synthase shows extensive functional analogy with LePGT1,
e.g. the preference for GPP as a substrate, which is recognized by
an aspartate-rich motif, and the requirement for divalent cations
with a preference for Mg2+ . By computational chemistry, the 3D
structure of LePGT1 was generated, to which two substrates,
GPP and PHB, were added by docking simulation (Figure 8A).
In a similar manner to FPP synthase, the diphosphate moiety
of GPP is recognized by two aspartate residues at positions 84
c The Authors Journal compilation c 2009 Biochemical Society
and 87 via two Mg2+ atoms through chelation bonds. The PHB
molecule is situated between the α-helix with Region I and that
containing Region III, where the carboxyl residue of PHB seems
to interact with another Mg2+ that is recognized by Asp208 and
Asp212 in Region III, whereas the orientation of PHB is so that
the m-position to be prenylated is facing the phosphate residue of
GPP. It is noteworthy that the distance between the OH residue
and the phosphate group is only 2.76 Å (1 Å = 0.1 nm). The
distance between the C1 atom of GPP and the carbon atom at
the m-position of PHB where the prenylation reaction takes place
is calculated to be 7.11 Å, which is larger than that of a soluble
type Streptomyces AS-PT, Orf2 (4 Å), whose crystal structure has
been characterized [22], but the distance of LePGT1 is similar
to that between the cysteine residue to be prenylated and the
C1 atom of FPP in human protein farnesyltransferase (7.3 Å)
[38]. This ‘bridge-like’ configuration of PHB between two αhelices containing either Region I and III, and the vicinity of
two substrates with reasonable orientations strongly suggests that
this model represents the structure of the substrate-binding site
of LePGT1. Actually, this model supports the biochemical data
using mutant enzymes demonstrated above with high conformity,
i.e. both Region I and III influence the recognition of each
substrate, and most amino acid residues (Asp84 , Asp87 and Asp91 )
involved in the catalytic functions in Region I as well as those in
Region III (Asp208 , Asp211 and Asp212 ) face the inside of the binding
pocket.
Using this model, it was calculated that the surface structure
of LePGT1-bound GPP, where the N-terminal region affecting
the substrate specificity (positions 41–170) is shown in cyan
(Figure 8B). This model demonstrates that the cavity where GPP
is bound provides a narrow space and the N-terminal region
takes the position close to the entrance of the cavity, suggesting
that the tight binding pocket formed by the N-terminal region is
the reason for the preference for GPP as the prenyl substrate.
In prenyltransferase proteins catalysing trans-prenyl-chainelongation such as FPP synthase, the aromatic amino acid
residue located at the fifth position upstream of the FARM
(DDxxD) is essential for controlling the product chain length
in trans prenyl chain elongation [39]. It was presumed that prenyl
substrate specificity for the chain length could also be regulated
in membrane-bound PPTs in a similar manner, but the substitution of the corresponding amino acids (Trp70 , Phe130 , Trp74 and
His140 ) of LePGT1 did not alter the substrate specificity (results not
shown). This suggests that the prenyltransferase activity for PHB
has a different regulatory mechanism from soluble-type prenyl
diphosphate synthases, and the molecular model of LePGT1
(Figure 8) indicates the specificity for the prenyl substrate to
be determined by the spatial size of the hydrophobic pocket in the
catalytic centre. This hypothesis is supported by the present study
of chimeric enzymes showing that specificity for chain length is
determined by the 130-amino acid segment of the N-terminus of
LePGT1, which is involved in the GPP-binding cavity.
The critical importance of the N-terminal region of PPT (e.g.
Ser41 to Ala170 of LePGT1) for the availability of the prenyl
substrates with different chain lengths was also demonstrated both
in the analysis of K m values and in the complementation study
with chimeric enzymes derived from LePGT1 and UBIA. The
physiological prenyl substrate of UBIA is octaprenyl diphosphate
(C40) in vivo, and UBIA showed a higher K m value for GPP
than did LePGT1. The K m value of Chimera 1 for GPP was
approximately equivalent to that of LePGT1, whereas Chimera
4 showed a much higher K m for GPP-like UBIA. It is worth
noting that in the yeast complementation study both Chimera 4
and UBIA could utilize hexaprenyl diphosphate (C30) in vivo as
a prenyl substrate, whereas LePGT1 and Chimera 1 could not.
Mutational analyses of membrane-bound prenyltransferase
Furthermore, the in vitro PHB farnesyltransferase assay showed
that Chimera 1 had LePGT1-type strict specificity for GPP but
Chimera 4 had relaxed specificity similar to UBIA. These results
indicate that the prenyl substrate specificity of membrane-type
PPTs is regulated by a different mechanism from that of soluble
prenyltransferases.
Plant prenyltransferases such as isoprene, monoterpene
sesquiterpene and diterpene synthases, which are soluble proteins,
require magnesium ions for their enzymatic activities, and their
common aspartate-rich motifs are highly conserved [40–42]. The
mechanism behind the requirement of Mg2+ for their catalytic
function was clearly demonstrated by X-ray crystallographic
studies. For instance, in FPP synthase, the catalytic centre
contains the exclusively conserved aspartate residues in FARM
and SARM (DDxxD), and the prenyl substrates were recognized
directly by these aspartate residues through the magnesium ions
in the catalytic centre of the enzyme [43].
The membrane-bound PPT family also needs magnesium ions
for enzymatic activity. These enzymes have two highly conserved
regions, i.e. Region I corresponding to FARM, where three
aspartates are completely conserved, and Region III, which also
contains three highly conserved aspartates but the amino acid
sequence is specific only to this family accepting PHB as an
aromatic substrate. Site-directed mutagenesis of LePGT1 showed
that only one conserved aspartate residue either in Region I or
in Region III acts concertedly in the catalytic centre of LePGT1
(Figure 2), perhaps in recognizing the diphosphate moiety of
the prenyl substrate via magnesium ions. This hypothesis seems
also to be valid for bacterial PPT, e.g. E. coli UBIA for UQ
biosynthesis [37]. Bacterial soluble-type AS-PTs that do not
require magnesium ions for their activity do not have these
aspartate-rich motifs [21], but these orthologues have not been
found in plants thus far.
The substitution of aspartates in Region I tended to have
severe negative effects on the expression of recombinant enzymes
(failure to express: D87N, D91E or D91N) or enzymatic activity
(no activity: D87E). However, substitution of the conserved
aspartates in Region III with glutamate (D208E and D212E)
resulted in detectable enzymatic activities with lower K m values
for GPP than the native LePGT1 (Figure 4), suggesting that these
glutamate residues partially complement the catalytic function in
maintaining the substrate-binding affinity. From these results, the
roles of conserved aspartates in the enzymatic function may differ
in a similar manner as in soluble trans-prenyl transferases [44]. In
the present 3D molecular model of LePGT1, Asp87 and Asp91 are
involved in the binding of prenyl diphosphate via magnesium ions
(Figure 8A). In addition, it is suggested in Figure 2 that Gln207 and
Asp211 also have critical roles in the binding of substrates or in
catalysing the reaction. Indeed, these amino acid residues, as well
as Asp208 and Asp212 , are located on the inner face of the substratebinding pocket and seem to be involved in the recognition of
both substrates (Figure 8A). Similarly, a contribution of Lys229 ,
located in Region III, to the enzymatic function, is predicted based
on the present mutational study (Figure 3) in which the alanine
mutant still displayed 12.9 % of the wild-type activity but the K m
values for both substrates strongly increased (approx. 3-fold). This
indicates that Lys229 is important for substrate recognition, and
the residue also faces the inside of the substrate-binding pocket.
Despite two limitations that the enzyme data is derived from the
in vitro assay using the microsomal system of transgenic yeast and
the structure is determined from molecular modelling based on a
soluble protein, reasonable explanations for the biochemical data
of LePGT1 can be drawn from the 3D structure shown in Figure 8.
Previously, Regions I and III were predicted to be responsible
for the binding of GPP and PHB respectively, due to sequence
239
similarity among various PPTs. However, the present mutational
analysis has clearly demonstrated that Regions I and III are
involved in the recognition of both substrates in a co-ordinated
manner, e.g. N83A and R76A in Region I caused remarkable
increases in the K m for PHB, whereas mutation of Ser219 and
Lys229 in Region III strongly affected the K m for GPP (Figure 3).
Although the structure is determined from molecular modelling
with FPP synthase as the template, the 3D model provides a
reasonable explanation of these biochemical findings, e.g. both
GPP and PHB are held by Regions I and III, where the distance
between the OH residue and phosphate group is 2.76 Å. It
also newly suggests that a magnesium ion is necessary for the
interaction between aspartate residues in Region III and the
carboxyl group of PHB (Figure 8A).
Recently, a 3D molecular model has been reported for the
UBIA protein, in which 5-epi-aristolochene synthase was used
as a template to generate the model [45]. A careful comparison
was made with the UBIA model and the present LePGT1 model,
especially at the active site of both enzymes, and some similar features were found, e.g. Asp87 (Asp71 of UBIA) and Asp91 (Asp75 of
UBIA) are involved in the binding of the diphosphate residue via a
magnesium ion. However, there are several discrepancies between
the UBIA model and the LePGT1 model and biochemical data
presented here. The most prominent difference involves Arg153
(Arg137 of UBIA), which was reportedly important for binding
prenyl diphosphate [45]. In the present study, R153A retained
strong enzymatic activity (81.6 % of the wild-type level), and thus
Arg153 does not have a critical role in catalytic function, whereas
the R153A mutant showed an influence on the K m value for PHB.
The weak activity of the R137A mutant of UBIA observed in
the previous study may not reflect the importance of this arginine
residue for enzymatic function, but may be due to a failure of the
expression of the recombinant protein in E. coli, which was not
monitored in the study [45]. Other minor differences were also
seen: Asp208 (Asp191 of UBIA) reportedly bound the OH group of
PHB, but is involved in recognition of the carboxyl group of PHB
in the present model; and Asp212 (Asp195 of UBIA) did not mediate
binding to the phosphate group, but recognition of PHB was
shown in the LePGT1 model. However, it should be emphasized
that the GPP-binding pattern is very similar between the two
models despite the usage of different templates. The configuration
of PHB in the binding pocket of LePGT1 is slightly different from
that in the UBIA model, in which Arg72 (UBIA) is responsible for
the interaction with the C-terminus of PHB [45], but this amino
acid is not conserved in other PPT members, e.g. this position is
glutamine in AtPPT1 and human COQ2, which is inconsistent
with their hypothesis concerning the role of the arginine
residue.
In the present study, the amino acid residues of LePGT1
critical for enzymatic activity and the region responsible
for the binding of substrates were elucidated biochemically.
These findings should improve the basic understanding of
the enzymatic mechanism of membrane-type AS-PTs. Other
membrane-bound AS-PT families in plants include homogentisate
prenyltransferases for vitamin E and plastoquinone biosynthesis
and also flavonoid prenyltransferases, which are responsible for
the production of many biologically active secondary metabolites
in plants. These membrane proteins have a similar membrane
topology to LePGT1 [46]. The molecular modelling method
applied to LePGT1 will also be applicable for these members
to understand their catalytic functions and will provide new
molecular engineering designs of membrane-bound AS-PTs
for the efficient production of prenylated aromatic compounds
using heterologous organisms such as bacteria or higher
plants.
c The Authors Journal compilation c 2009 Biochemical Society
240
K. Ohara and others
AUTHOR CONTRIBUTION
Kazufumi Yazaki and Nobuhiro Fukushima designed research; Kazuaki Ohara and Ayumu
Muroya performed research; Kazuaki Ohara, Ayumu Muroya, Nobuhiro Fukushima and
Kazufumi Yazaki analysed data and Kazuaki Ohara, Ayumu Muroya and Kazufumi Yazaki
wrote the paper.
ACKNOWLEDGEMENTS
The yeast shuttle vector pDR196 was a generous gift from Dr Wolf Frommer (Department
of Plant Biology, Carnegie Institution for Science, Stanford, CA, U.S.A.). We also thank
Dr Lutz Heide (Pharmaceutical Institute, University of Tübingen, Germany) for cDNA of
the UBIA gene. We are grateful to Dr Toshiaki Umezawa of Kyoto University for LC-MS
analyses. The analysis of DNA sequences was conducted by the Life Research Support
Center of Akita Prefectural University. We thank Accelrys (San Diego, CA, U.S.A.) for
approval to use the Accelrys Discovery Studio in the modelling.
FUNDING
This work was supported in part by a Grant-in-aid for Scientific Research [grant numbers
17310126 and 21310141 (to K. Y.)], a Grant from the Research for the Future Program:
‘Molecular mechanisms on regulation of morphogenesis and metabolism leading to
increased plant productivity’ [grant number 00L01605 (to K. Y.)] of the Ministry of
Education, Culture, Sports, Science and Technology of Japan, and by a Research
Fellowship from the Japan Society for the Promotion of Science for Young Scientists
[grant number 17·2011 (to K. O.)].
REFERENCES
1 Kawamukai, M. (2002) Biosynthesis, bioproduction and novel roles of ubiquinone.
J. Biosci. Bioeng. 94, 511–517
2 Swiezewska, E., Dallner, G., Andersson, B. and Ernster, L. (1993) Biosynthesis of
ubiquinone and plastoquinone in the endoplasmic reticulum-Golgi membranes of
spinach leaves. J. Biol. Chem. 268, 1494–1499
3 Sadre, R., Gruber, J. and Frentzen, M. (2006) Characterization of homogentisate
prenyltransferases involved in plastoquinone-9 and tocochromanol biosynthesis.
FEBS Lett. 580, 5357–5362
4 Collakova, E. and DellaPenna, D. (2001) Isolation and functional analysis of
homogentisate phytyltransferase from Synechocystis sp. PCC 6803 and Arabidopsis .
Plant Physiol. 127, 1113–1124
5 Schledz, M., Seidler, A., Beyer, P. and Neuhaus, G. (2001) A novel phytyltransferase from
Synechocystis sp. PCC 6803 involved in tocopherol biosynthesis. FEBS Lett. 499, 15–20
6 Schroder, G., Zahringer, U., Heller, W., Ebel, J. and Grisebach, H. (1979) Biosynthesis of
antifungal isoflavonoids in Lupinus albus . Enzymatic prenylation of genistein and
2 -hydroxygenistein. Arch. Biochem. Biophys. 194, 635–636
7 Yamamoto, H., Senda, M. and Inoue, K. (2000) Flavanone 8-dimethylallyltransferase in
Sophora flavescens cell suspension cultures. Phytochemistry 54, 649–655
8 Zhao, P., Inoue, K., Kouno, I. and Yamamoto, H. (2003) Characterization of leachianone G
2”-dimethylallyltransferase, a novel prenyl side-chain elongation enzyme for the
formation of the lavandulyl group of sophoraflavanone G in Sophora flavescens Ait. cell
suspension cultures. Plant Physiol. 133, 1306–1313
9 Biggs, D. R., Welle, R., Visser, F. R. and Grisebach, H. (1987)
Dimethylallylpyrophosphate:3,9-dihydroxypterocarpan 10-dimethylallyl transferase from
Phaseolus vulgaris . Identification of the reaction product and properties of the enzyme.
FEBS Lett. 220, 223–226
10 Welle, R. and Grisebach, H. (1991) Properties and solubilization of the prenyltransferase
of isoflavonoid phytoalexin biosynthesis in soybean. Phytochemistry 30, 479–484
11 Laflamme, P., Khouri, H., Gulick, P. and Ibrahim, R. (1993) Enzymatic prenylation of
isoflavones in white lupin. Phytochemistry 34, 147–151
12 Dhillon, D. S. and Brown, S. A. (1976) Localization, purification, and characterization of
dimethylallylpyrophosphate:umbelliferone dimethylallyltransferase from Ruta graveolens .
Arch. Biochem. Biophys. 177, 74–83
13 Hamerski, D., Schmitt, D. and Matern, U. (1990) Induction of two prenyltransferases for
the accumulation of coumarin phytoalexins in elicitor-treated Ammi majus cell
suspension cultures. Phytochemistry 29, 1131–1135
14 Zuurbier, K. W. M., Fung, S. Y., Scheffer, J. J. C. and Verpoorte, R. (1998) In-vitro
prenylation of aromatic intermediates in the biosynthesis of bitter acids in Humulus
lupulus . Phytochemistry 49, 2315–2322
15 Fellermeier, M. and Zenk, M. H. (1998) Prenylation of olivetolate by a hemp transferase
yields cannabigerolic acid, the precursor of tetrahydrocannabinol. FEBS Lett. 427,
283–285
c The Authors Journal compilation c 2009 Biochemical Society
16 Tahara, S. and Ibrahim, R. K. (1995) Prenylated isoflavonoids-an update. Phytochemistry
38, 1073–1094
17 Morandi, D. (1996) Occurrence of phytoalexins and phenolic compounds in
endomycorrhizal interactions, and their potential role in biological control. Plant Soil
185, 241–251
18 Wang, B. H., Ternai, B. and Polya, G. (1997) Specific inhibition of cyclic AMP-dependent
protein kinase by warangalone and robustic acid. Phytochemistry 44, 787–796
19 Henderson, M. C., Miranda, C. L., Stevens, J. F., Deinzer, M. L. and Buhler, D. R. (2000)
In vitro inhibition of human P450 enzymes by prenylated flavonoids from hops, Humulus
lupulus . Xenobiotica 30, 235–251
20 Di Pietro, A., Conseil, G., Perez-Victoria, J. M., Dayan, G., Baubichon-Cortay, H.,
Trompier, D., Steinfels, E., Jault, J. M., de Wet, H., Maitrejean, M., et al. (2002)
Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and
related ABC transporters. Cell. Mol. Life Sci. 59, 307–322
21 Pojer, F., Wemakor, E., Kammerer, B., Chen, H., Walsh, C. T., Li, S. M. and Heide, L.
(2003) CloQ, a prenyltransferase involved in clorobiocin biosynthesis. Proc. Natl. Acad.
Sci. U.S.A. 100, 2316–2321
22 Kuzuyama, T., Noel, J. P. and Richard, S. B. (2005) Structural basis for the promiscuous
biosynthetic prenylation of aromatic natural products. Nature 435, 983–987
23 Siebert, M., Bechthold, A., Melzer, M., May, U., Berger, U., Schroder, G., Schroder, J.,
Severin, K. and Heide, L. (1992) Ubiquinone biosynthesis. Cloning of the genes coding
for chorismate pyruvate-lyase and 4-hydroxybenzoate octaprenyl transferase from
Escherichia coli . FEBS Lett. 307, 347–350
24 Ashby, M. N., Kutsunai, S. Y., Ackerman, S., Tzagoloff, A. and Edwards, P. A. (1992)
COQ2 is a candidate for the structural gene encoding para -hydroxybenzoate:
polyprenyltransferase. J. Biol. Chem. 267, 4128–4136
25 Suzuki, K., Ueda, M., Yuasa, M., Nakagawa, T., Kawamukai, M. and Matsuda, H. (1994)
Evidence that Escherichia coli ubiA product is a functional homolog of yeast COQ2, and
the regulation of ubiA gene expression. Biosci. Biotechnol. Biochem. 58, 1814–1819
26 Okada, K., Ohara, K., Yazaki, K., Nozaki, K., Uchida, N., Kawamukai, M., Nojiri, H. and
Yamane, H. (2004) The AtPPT1 gene encoding 4-hydroxybenzoate polyprenyl
diphosphate transferase in ubiquinone biosynthesis is required for embryo development
in Arabidopsis thaliana . Plant Mol. Biol. 55, 567–577
27 Ohara, K., Yamamoto, K., Hamamoto, M., Sasaki, K. and Yazaki, K. (2006) Functional
characterization of OsPPT1, which encodes p -hydroxybenzoate polyprenyltransferase
involved in ubiquinone biosynthesis in Oryza sativa . Plant Cell Physiol. 47, 581–590
28 Forsgren, M., Attersand, A., Lake, S., Grunler, J., Swiezewska, E., Dallner, G. and Climent,
I. (2004) Isolation and functional expression of human COQ2 , a gene encoding a
polyprenyl transferase involved in the synthesis of CoQ. Biochem. J. 382, 519–526
29 Ohara, K., Kokado, Y., Yamamoto, H., Sato, F. and Yazaki, K. (2004) Engineering of
ubiquinone biosynthesis using the yeast coq2 gene confers oxidative stress tolerance in
transgenic tobacco. Plant J. 40, 734–743
30 Yazaki, K., Kunihisa, M., Fujisaki, T. and Sato, F. (2002) Geranyl diphosphate:4hydroxybenzoate geranyltransferase from Lithospermum erythrorhizon . Cloning and
characterization of a key enzyme in shikonin biosynthesis. J. Biol. Chem. 277,
6240–6246
31 Heide, L. and Berger, U. (1989) Partial purification and properties of geranyl
pyrophosphate synthase from Lithospermum erythrorhizon cell cultures. Arch. Biochem.
Biophys. 273, 331–338
32 Melzer, M. and Heide, L. (1994) Characterization of polyprenyldiphosphate:
4-hydroxybenzoate polyprenyltransferase from Escherichia coli . Biochim. Biophys. Acta
1212, 93–102
33 Rentsch, D., Laloi, M., Rouhara, I., Schmelzer, E., Delrot, S. and Frommer, W. B. (1995)
NTR1 encodes a high affinity oligopeptide transporter in Arabidopsis . FEBS Lett. 370,
264–268
34 Uchida, N., Suzuki, K., Saiki, R., Kainou, T., Tanaka, K., Matsuda, H. and Kawamukai, M.
(2000) Phenotypes of fission yeast defective in ubiquinone production due to disruption
of the gene for p -hydroxybenzoate polyprenyl diphosphate transferase. J. Bacteriol. 182,
6933–6939
35 Shitan, N., Bazin, I., Dan, K., Obata, K., Kigawa, K., Ueda, K., Sato, F., Forestier, C. and
Yazaki, K. (2003) Involvement of CjMDR1, a plant multidrug-resistance-type ATP-binding
cassette protein, in alkaloid transport in Coptis japonica . Proc. Natl. Acad. Sci. U.S.A.
100, 751–756
36 Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,
248–254
37 Brauer, L., Brandt, W. and Wessjohann, L. A. (2004) Modeling the E. coli
4-hydroxybenzoic acid oligoprenyltransferase (ubiA transferase) and characterization of
potential active sites. J. Mol. Model. 10, 317–327
38 Long, S. B., Casey, P. J. and Beese, L. S. (2002) Reaction path of protein
farnesyltransferase at atomic resolution. Nature 419, 645–650
Mutational analyses of membrane-bound prenyltransferase
39 Ohnuma, S., Narita, K., Nakazawa, T., Ishida, C., Takeuchi, Y., Ohto, C. and Nishino, T.
(1996) A role of the amino acid residue located on the fifth position before the first
aspartate-rich motif of farnesyl diphosphate synthase on determination of the final
product. J. Biol. Chem. 271, 30748–30754
40 Miller, B., Oschinski, C. and Zimmer, W. (2001) First isolation of an isoprene synthase
gene from poplar and successful expression of the gene in Escherichia coli . Planta 213,
483–487
41 Sasaki, K., Ohara, K. and Yazaki, K. (2005) Gene expression and characterization of
isoprene synthase from Populus alba . FEBS Lett. 579, 2514–2518
42 Bohlmann, J., Meyer-Gauen, G. and Croteau, R. (1998) Plant terpenoid synthases:
molecular biology and phylogenetic analysis. Proc. Natl. Acad. Sci. U.S.A. 95,
4126–4133
241
43 Marrero, P. F., Poulter, C. D. and Edwards, P. A. (1992) Effects of site-directed
mutagenesis of the highly conserved aspartate residues in domain II of farnesyl
diphosphate synthase activity. J. Biol. Chem. 267, 21873–21878
44 Song, L. and Poulter, C. D. (1994) Yeast farnesyl-diphosphate synthase: site-directed
mutagenesis of residues in highly conserved prenyltransferase domains I and II. Proc.
Natl. Acad. Sci. U.S.A. 91, 3044–3048
45 Brauer, L., Brandt, W., Schulze, D., Zakharova, S. and Wessjohann, L. (2008) A structural
model of the membrane-bound aromatic prenyltransferase UbiA from. E. coli.
Chembiochem 9, 982–992
46 Sasaki, K., Mito, K., Ohara, K., Yamamoto, H. and Yazaki, K. (2008) Cloning and
characterization of naringenin 8-prenyltransferase, a flavonoid-specific prenyltransferase
of Sophora flavescens . Plant Physiol. 146, 1075–1084
Received 30 September 2008/21 April 2009; accepted 24 April 2009
Published as BJ Immediate Publication 24 April 2009, doi:10.1042/BJ20081968
c The Authors Journal compilation c 2009 Biochemical Society
Biochem. J. (2009) 421, 231–241 (Printed in Great Britain)
doi:10.1042/BJ20081968
SUPPLEMENTARY ONLINE DATA
Functional characterization of LePGT1, a membrane-bound
prenyltransferase involved in the geranylation of p -hydroxybenzoic acid
Kazuaki OHARA*1 , Ayumu MUROYA†, Nobuhiro FUKUSHIMA† and Kazufumi YAZAKI*2
*Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University, Uji 611-0011, Japan, and †Science & Technology Systems, Inc., 1-20-1
Shibuya, Tokyo 150-0002, Japan
Figure S1
Oligonucleotides used in the present study
Mutagenic oligonucleotides designed to produce the desired point mutations are listed; underlining indicates changed codons for mutagenesis.
1
Present address: Central Laboratories for Frontier Technology, Kirin Holdings Co., Ltd., 1-13-5 Fukuura Kanazawa-ku, Yokohama-shi, Kanagawa
236-0004, Japan.
2
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2009 Biochemical Society
K. Ohara and others
Figure S2
Prediction of transmembrane domains of LePGT1
Predicted transmembrane domains (TM) are underlined, and the three conserved regions,
Region I, II and III, are indicated with red lines. Predictions were carried out using the following
programs; HMMTOP (http://www.enzim.hu/hmmtop/), TMHMM (http://www.cbs.dtu.dk/services/TMHMM/),
TMPRED
(http://www.ch.embnet.org/software/TMPRED_form.html),
CONPRE (Conpred2; http://bioinfo.si.hirosaki-u.ac.jp/∼ConPred2/) and MEMSAT (http://saier144-37.ucsd.edu/memsat.html). H, α-helix; i, inside; o, outside.
Received 30 September 2008/21 April 2009; accepted 24 April 2009
Published as BJ Immediate Publication 24 April 2009, doi:10.1042/BJ20081968
c The Authors Journal compilation c 2009 Biochemical Society