Báo cáo khoa học: Molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis A key factor participating in natural rubber biosynthesis pdf

Molecular cloning, expression and characterization of cDNA
encoding
cis
-prenyltransferases from
Hevea brasiliensis
A key factor participating in natural rubber biosynthesis
Kasem Asawatreratanakul
1,2
, Yuan-Wei Zhang
1,
*, Dhirayos Wititsuwannakul
3
, Rapepun Wititsuwannakul
4
,
Seiji Takahashi
1
, Atiya Rattanapittayaporn
4
and Tanetoshi Koyama
1
1
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan;
2
Department of Chemistry,
Thaksin University, Songkla, Thailand;
3
Department of Biochemistry, Mahidol University, Bangkok, Thailand;
4
Department of
Biochemistry, Prince of Songkla University, Hat-Yai, Thailand

Natural rubber from Hevea brasiliensis is a high molecular
mass polymer of isoprene units with cis-configuration. The
enzyme responsible for the cis-1,4-polymerization of iso-
prene units has been identified as a particle-bound rubber
transferase, but no gene encoding this enzyme has been
cloned from rubber-producing plants. By using sequence
information from the conserved regions of cis-prenyl chain
elongating enzymes that were cloned recently, we have iso-
lated and characterized cDNAs from H. brasiliensis for a
functional factor participating in natural rubber biosyn-
thesis. Sequence analysis revealed that all of the five highly
conserved regions among cis-prenyl chain elongating
enzymes were found in the protein sequences of the Hevea
cis-prenyltransferase. Northern blot analysis indicated that
the transcript(s) of the Hevea cis-prenyltransferase were
expressed predominantly in the latex as compared with other
Hevea tissues examined. In vitro rubber transferase assays
using the recombinant gene product overexpressed in
Escherichia coli revealed that the enzyme catalyzed the for-
mation of long chain polyprenyl products with approximate
sizes of 2 · 10
3
)1 · 10
4
Da. Moreover, in the presence of
washed bottom fraction particles from latex, the rubber
transferase activity producing rubber product of high
molecular size was increased. These results suggest that the
Hevea cis-prenyltransferase might require certain activation
factors in the washed bottom fraction particles for the pro-

duction of high molecular mass rubber.
Keywords: prenyltransferase; rubber transferase; Hevea
brasiliensis; isoprenoid.
Although over 2000 species of higher plants are recognized
for producing latex with polyisoprenes [1], only the rubber
tree (Hevea brasiliensis) has been established as a key
commercial rubber source due to its good yield of rubber
and the excellent physical properties of the rubber products.
Hevea rubber is a high molecular mass polymer of isoprene
units in cis-configuration. Rubber molecules are produced
and aggregated or packaged as rubber particles in latex
vessels of the rubber tree [2]. Although natural rubber is
synthesized and made almost entirely of isoprene units
derived from isopentenyl diphosphate (IPP), an allylic
diphosphate is also required as the priming cosubstrate to
initiate the subsequent extensive prenyl chain elongation
process for the formation of rubber macromolecules [3–5].
Synthesis of the allylic prenyl diphosphates are catalyzed
by IPP isomerase and trans-prenyltransferase enzymes, the
enzymatic activities of which were found in both the bottom
fraction and the supernatant cytosol (C-serum) of centri-
fuged fresh Hevea latex [6–8].
The enzyme responsible for cis-1,4-polymerization of
isoprene units from IPP onto the allylic primer has
been identified as a particle-bound rubber transferase
(EC 2.5.1.20) [3,9,10]. The particle-bound rubber transferase
activities were demonstrated in various rubber producing
plants; guayule [4,5], Ficus elastica [11] and Ficus carica [12].
It has been shown that IPP is incorporated into rubber at
the surface of the rubber particles in latex, by reaction with a

terminal allylic diphosphate group of the rubber molecules
[13–15]. This indicates that rubber transferase is bound to
the rubber particles and is still present even after the
particles are washed repeatedly. However, the precise
mechanism for the biosynthesis of rubber molecules has
not yet been established. Moreover, the exact site of the
formation of new rubber molecules still remains unknown.
It has been suggested that the bottom fraction membrane
could possibly serve as the site for initiation of new rubber
formation [16,17].
Correspondence to T. Koyama, Institute of Multidisciplinary Research
for Advanced Materials, Tohoku University, Katahira 2-1-1,
Aoba-ku, Sendai 980–8577, Japan.
Fax: + 81 22 217 5620, Tel.: + 81 22 217 5621,
E-mail:
Abbreviations: IPP, isopentenyl diphosphate; HRT, Hevea rubber
transferase; WBP, washed bottom fraction particles; IPTG, isopropyl
thio-b-
D
-galactoside; GPC, gel permeation chromatography;
dedol-PP, dehydrodolichyl diphosphate.
Enzyme: rubber cis-polyprenylcistransferase (EC 2.5.1.20).
*Present address: Department of Pharmacology, School of Medicine,
Yale University, New Haven, CT, USA.
Note: The nucleotide sequences reported in this paper are available in
the DDBJ/GenBank
TM
/EMBL Data Bank under the accession
numbers AB061234 and AB064661.
(Received 15 July 2003, revised 26 September 2003,

accepted 2 October 2003)
Eur. J. Biochem. 270, 4671–4680 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03863.x
In the biosynthesis of polyisoprenoid compounds, the
prenyl chain elongation, catalyzed by prenyltransferases,
proceeds consecutively and terminates precisely at certain
chain lengths according to the specificities of individual
enzymes [18–20]. These enzymes have been classified into
two major groups, referred to as trans-or(E)-prenyl
diphosphate synthases (trans-prenyltransferases) and as
cis-or(Z)-prenyl diphosphate synthases (cis-prenyltrans-
ferases), depending on the stereochemistry of the conden-
sation reaction of IPP with the corresponding allylic prenyl
diphosphate initiator. During the past 16 years, many
different genes encoding trans-prenyltransferases have been
cloned and characterized [19,20]. On the other hand, very
limited information was available on cis-prenyltransferases
until the recent cloning and characterization of the genes
encoding cis-prenyltransferases from Micrococcus luteus
B-P 26, Escherichia coli, Haemophilus influenzae, Strepto-
coccus pneumonica, Saccharomyces cerevisiae and Arabid-
opsis thaliana [21–25].
Natural rubber has been thought to be made almost
entirely of cis-isoprene units derived from IPP, and the
enzyme responsible for polymerization is believed to have
characteristics similar to the cis-prenyl diphosphate synth-
ases. However, the genes encoding Hevea cis-prenyltrans-
ferases or rubber transferase (HRT) from H. brasiliensis
have not yet been reported. It was suggested that rubber
biosynthesis in H. brasiliensis is mediated by the association
of a soluble trans-prenyltransferase with a rubber elongation

factor, a 14.6 kDa protein, tightly bound to the rubber
particles in the laticifers [26]. However, Cornish [10] has
demonstrated that the soluble trans-prenyltransferase func-
tions as farnesyl diphosphate synthase, and almost certainly
plays no direct role in the cis-1,4-polyisoprene elongation.
A detailed understanding of rubber biosynthesis proces-
ses at the molecular level is important for genetic manipu-
lation of the isoprenoid biosynthesis pathway enzymes. In
this study, by using the sequence information from the
conserved regions of the cis-prenyl chain elongating
enzymes [20–22], we isolated and characterized two Hevea
cis-prenyltransferases cDNAs designated as HRT1 and
HRT2 from the H. brasiliensis latex. Sequence analysis
showed that all five regions that are conserved among
cis-prenyl chain elongating enzymes were present in each of
the deduced amino acid sequences encoded by the two
cDNAs. In vitro assay of the rubber transferase activity of
the recombinant HRT proteins was carried out in the
presence of washed bottom fraction particles (WBP) of fresh
Hevea latex. These results suggest that the HRT2 protein
catalyzes the synthesis of new rubber molecules with the
activeinvolvementofanumberoffactorsinWBPoffresh
Hevea latex.
Materials and methods
Plant materials and RNA isolation
Latex and various tissue samples were obtained from ten-
year old rubber plants (H. brasiliensis clone RRIM 600)
being grown at the Rubber Research Center of Songkla,
Thailand. Latex collection was performed as described by
Kush et al. [27]. The latex total RNA was extracted by

using RNAgents Total RNA Isolation System (Promega).
Total RNAs of rubber leaf and other tissues were
obtained using QuickPrep Total RNA Extraction kit
(Amersham Biosciences) and RNA Isolation kit (Qiagen),
respectively. Poly(A)
+
RNA was isolated from the total
RNA with Oligodex-dT30 mRNA Purification kit
(TaKaRa, Ohtsu, Japan).
RT-PCR amplification of
Hevea cis
-prenyltransferase
cDNA fragment
RT-PCR was carried out using Ready-To-Go
TM
RT-PCR
Beads (Amersham Biosciences). First strand cDNA syn-
thesis was performed by reverse transcription with 100 ng of
poly(A)
+
RNA isolated from latex using poly-d(T)
12)18
primer. Two oligonucleotide degenerate primers were
designed to amplify the Hevea cis-prenyltransferase cDNA
fragment according to the highly conserved regions among
cis-prenyl chain elongating enzymes; sense primer, P1
(AFIMDGN, region I) 5¢-GCTTTTATTATGGAYG
GHAA-3¢ and antisense primer, P2 (IRTSGE, region V)
5¢-CTCACCAGAWGTWCKWAT-3¢,whereHisA,Cor
T; K is G or T; W is A or T and Y is C or T. PCR was

performed in a final volume of 50 lL containing 50 pmol of
amplification primer pair for 45 cycles of 30 s at 95 °C, 30 s
at 45 °C and 1 min at 72 °C with a 5 min preheat and a
10 min final extension at 72 °C. The resulting band of PCR
products were extracted from agarose gel and subcloned
into pT7Blue T vector (Novagen) for sequencing. One
of the resulting clones, which showed homology to
cis-prenyltransferases, was termed LT600.
3¢- and 5¢-RACE reaction and cloning of
Hevea
cis
-prenyltransferase cDNA
Cloning of the full-length cDNA of Hevea cis-prenyltrans-
ferase, used poly(A)
+
RNA from latex as the template for
performing both 3¢-and5¢-RACE reactions based on the
cDNA sequence of LT600. The procedures applied for
3¢-and5¢-RACE reaction were according to the manufac-
turers instructions (3¢-Full RACE Core Set, TaKaRa;
5¢-RACE kit, Roche). The primer, F1, used for 3¢-RACE
was 5¢-AGGGCTACTGCCAACAATTCC-3¢ and the
primers, R1 and R2, used for 5¢-RACE reaction were
5¢-GCTTCCAGTTGCATTTGCCTCCTCC-3¢ and 5¢-GC
TAAAGGCATAGATAGTCGC-3¢ respectively. Accord-
ing to the sequence information obtained by the 5¢-or
3¢-RACE reaction, the cDNA was amplified by RT-PCR
with the latex poly(A)
+
RNA as the template and then

sequenced. Finally, two cDNAs were obtained and desig-
nated HRT1 and HRT2, respectively.
DNA sequencing analysis
Sequencing reactions were performed using Thermo
Sequenase Cycle Sequencing kit (Amersham Pharmacia
Biotech) with fluorescent labeled primers. Nucleotide
sequences were determined by the dideoxy chain termin-
ation method [28] with a DNA sequencer (LI-COR,
model 4200, LI-COR Inc., Lincoln, NE, USA). Compu-
ter analysis and comparison of DNA sequences were
carried out using
GENETYX
genetic information processing
software (Genetyx Corp., Tokyo, Japan).
4672 K. Asawatreratanakul et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Analysis of
HRT
gene expression
For Northern hybridization, total RNAs (15 lg) from
various tissues were subjected to electrophoresis on 0.8%
agarose gel containing 1% formaldehyde, and blotted onto
a positively charged nylon membrane (Roche). The mem-
branes were hybridized with
32
P-labeled HRT cDNAs for
1h at 68°C in ExpressHyb solution (Clontech). High
stringency washes were performed twice at 50 °Cin
2 · NaCl/Cit, 0.05% SDS and twice in 0.1 · NaCl/Cit,
0.1% SDS. The hybridized membranes were exposed for
12 h on a Fuji imaging plate and then analyzed with a Fuji

BAS 1000 Mac Bioimage analyzer. RT-PCR for the
analysis of HRT expression was performed by using total
RNAs (2 lg) from various Hevea tissues, amplified with
HRT1 or HRT2 specific primers. The PCR reaction was
carried out with 25 cycles of programmed temperature
control of 30 s at 95 °C, 30 s at 50 °C and 1 min at 72 °C
with a 5 min preheat at 95 °C and a 10 min final extension
at 72 °C using primers, S1 (5¢-GCAAATGCAACTGGA
AGCGG-3¢)andA1(5¢-ACAGCCTGCTAGCAAAGA
GG-3¢) for amplification of HRT1, and primers S2
(5¢-GAAGAATCCTCTAAGGATAA-3¢)andA2(5¢-TA
CAAGGATTAATCCCTTGC-3¢) for amplification of
HRT2. The PCR products were analyzed by agarose gel
electrophoresis with ethidium bromide staining.
Construction of expression vector systems in
E. coli
,
and purification of
Hevea cis
-prenyltransferase
Expression vector systems for the HRTs were constructed
using pET32b(+) vector (Novagen), which is designed to
express the gene product as a thioredoxin- and His-tagged
fusion protein, suitable for production of soluble protein in
E. coli cytoplasm and rapid purification. The restriction
enzyme recognition site for NcoIorBamHI was introduced
by PCR at either the 5¢-end or 3¢-end of the coding regions
of the two HRT cDNAs. The resulting fragments were
sequenced, digested with NcoIandBamHI, and ligated into
the NcoI–BamHI vector of pET32b(+), yielding the

expression plasmids pETHRT1 and pETHRT2. Each of
the expression plasmids was used for transformation of
E. coli BL21(DE3), and 1 mL of an overnight culture
of the transformant in Luria–Bertani medium containing
50 lgÆmL
)1
ampicillin was inoculated into 200 mL of
M9YG medium [29] containing 50 lgÆmL
)1
ampicillin.
The cells were grown at 37 °CtoanA
600
value of 0.4.
Isopropyl thio-b-
D
-galactoside (IPTG) was added to a final
concentration of 0.5 m
M
, and then a further incubation at
30 °C for 4 h was carried out. Overproduction of the
proteins was confirmed by SDS/PAGE according to the
standard method of Laemmli [30].
The cells were harvested by centrifugation (5000 g for
10 min) and then disrupted by sonication. The cell homo-
genates were fractionated into soluble and insoluble proteins
by centrifugation at 8000 g for 10 min. The expressed
proteins were purified essentially according to the protocol
of Xpress Protein Purification System (Invitrogen), using
aNi
2+

nitrilotriacetic acid-agarose column. The soluble
proteins were applied to a Ni
2+
resin column, and the His-
tagged fusion protein was eluted with a gradient of
50–500 m
M
imidazole in 20 m
M
phosphate buffer, pH 6.0.
The insoluble proteins were solubilized with 6
M
guanidine
hydrochloride lysis buffer, pH 7.8 and subjected to a Ni
2+
resin column. The column was washed and the tagged
protein was eluted with 8
M
urea in 20 m
M
phosphate
buffer, pH 4.0. The purified protein was renaturated by
removal of urea via stepwise dialysis. The portion of purified
fusion protein was treated with enterokinase to remove the
N-terminal fused thioredoxin. The digested protein was
used for the rubber transferase activity assay and for
product analysis. Protein concentration was measured by
the Bradford method [31].
In vitro
rubber transferase activity assay

The washed bottom fraction particles (WBP) were prepared
by ultracentrifugation (49 000 g,45min,4°C) of fresh
Hevea latex followed by repeated washing of the fresh
bottom fraction with 50 m
M
Tris/HCl buffer, pH 7.4
containing 0.9% NaCl (w/v) according to the method of
Wititsuwannakul et al. [17]. The rubber transferase activity
assay was performed by the modified method of Tangpak-
dee et al. [16]. The reaction mixture contained, in a final
volume of 0.2 mL, 50 m
M
Tris/HCl buffer (pH 7.4), 30 m
M
KCl, 2 m
M
MgCl
2
,5l
M
ZnCl
2
,5m
M
dithiothreitol,
20 m
M
KF, 0.1 m
M
deoxycholate, 0.5 mg of WBP, 15 l

M
farnesyl diphosphate, 50 l
M
[1-
14
C]IPP (2.15 GBqÆmmol
)1
,
Amersham Pharmacia Biotech), and a suitable amount of
cell-free homogenate or purified protein. After incubation
for 4 h at 30 °C, short and medium chain polyprenyl
diphosphate products were extracted with 1-butanol, and
then residual radioactive rubber in aqueous phase was
extracted three times with 0.6 mL of toluene/hexane
mixture (1 : 1 v/v). The extracts were concentrated to a
small volume and the radioactivity was measured with an
Aloka LSC-1000 liquid scintillation counter (Tokyo,
Japan). The rubber transferase activity was determined by
measuring the amount of [
14
C]IPP incorporated into rubber
in the toluene/hexane extracts.
Analysis of HRT Reaction Products
The radioactive products extracted with the toluene/hexane
mixture were treated with potato acid phosphatase accord-
ing to the method reported previously [32]. The radioactive
products were extracted with toluene and analyzed by TLC
on a reversed phase RP-18 plate (Merck) with a solvent
system of acetone/water (39 : 1 v/v). The positions of
authentic standards were visualized with iodine vapor, and

the distribution of radioactive products on the TLC plate
was analyzed with a Fuji BAS-1000 Mac Bioimage
analyzer.
Distribution of molecular size of rubber products were
analyzed by gel permeation chromatography (GPC) which
was carried out with a Tosoh high performance liquid
chromatography system, equipped in tandem with a series
of four TSK gel GPC columns, G7000H, G5000H,
G2500H, and G1000H (Tosoh Corp., Tokyo, Japan), each
of which has an exclusion limit of 4 · 10
8
,4· 10
6
,2· 10
4
and 1 · 10
3
Da, respectively. The chromatography was
carried out at 35 °C using tetrahydrofuran as eluent, at a
flow rate of 0.5 mLÆmin
)1
. The eluate was monitored by
UV absorption at 210 nm following collection at 1 min
Ó FEBS 2003 cis-Prenyltransferase from Hevea brasiliensis (Eur. J. Biochem. 270) 4673
intervals, and assayed for radioactivity. The molecular mass
of the reaction products were estimated by comparing them
with the elution volumes of commercially available standard
polystyrenes.
Expression of HRT in yeast strain SNH23-7D
To express the HRT cDNA in the yeast mutant strain

SNH23-7D (MATa rer2-2 mfa1::ADE2 mfa2::TRP1
bar1::HIS3ade2trp1his3leu2ura3lys2) according to the
study of Sato et al.[23],theBglII–SalI fragments containing
HRT1 or HRT2 cDNA were cloned into the corresponding
sites of plasmid pJR1133, which contain the URA3 marker
gene and yeast glyceraldehyde phosphate dehydrogenase
promoter [25]. The resulting plasmids designated pJRHRT1
and pJRHRT2, contained HRT1 and HRT2 respectively.
The SNH23-7D yeast strain was transformed with plasmid
pJRHRT1 and pJRHRT2 according to the protocol of
Fast
TM
Yeast Transformation kit (Geno Technology Inc.,
St. Louis, USA). Ura
+
transformants were selected at
23 °C on agar plates containing minimal medium [0.67%
(w/v) yeast nitrogen base without amino acid, 2% glucose,
supplemented with 60 lgÆmL
)1
leucine and 30 lgÆmL
)1
lysine]. Selected Ura
+
colonies and yeast strain SNY9
(MATa mfa1::ADE2 mfa2::TRP1 bar1::HIS3 ade2 trp1
his3 leu2 ura3 lys2), which contains the wild type RER2
gene, were streaked on agar plates containing YPD medium
and incubated at 23 °Cor37°C.
Results

Isolation and characterization of HRT cDNAs
A pair of degenerate primers designed from two highly
conserved regions (I and V) of the known cis-prenyl chain
elongating enzymes [20–22,33–35], were used to amplify of a
possible cDNA encoding cis-prenyltransferase(s) in Hevea
latex that might be responsible for the prenyl chain
elongation of natural rubber. RT-PCR with these primers
yielded amplified products of  600 bp in length. These
products were extracted and cloned into pT7Blue T vector.
One of the cDNA fragments, which contained regions
homologous to those of the conserved regions II, III and IV
of cis-prenyltransferase was designated as LT600. The
Fig. 1. Nucleotide and deduced amino acid sequences of HRT1 (A) and HRT2 (B). Numbers of nucleotide sequence and amino acid sequence are
indicated on the left and right, respectively. The underline in HRT1 indicates the sequence corresponding to the LT600 fragments.
4674 K. Asawatreratanakul et al. (Eur. J. Biochem. 270) Ó FEBS 2003
nucleotide sequence of the fragment was used to design the
primers to amplify unknown 3¢-and5¢-end sequences of
HRT cDNAs by using 3¢-and5¢-RACE strategies. The
amplified products of the 3¢-end gave two distinct cDNA
fragments which were 689 and 557 bp in length, with a
sequence identity of 85% having identical sequence near the
3¢-end of the possible open reading frame. When the
5¢-cDNA was amplified, only a 424 bp cDNA fragment was
obtained. To obtain full-length sequences of these ORFs, a
pair of primers was designed according to the sequence
information from the RACE analysis. After amplification
by RT-PCR from latex poly(A)
+
RNA, we sequenced a
number of fragments and obtained two distinct cDNAs that

contain sequences identical to those obtained by 3¢-RACE.
General cDNAs containing 5¢-and3¢-untranslated regions
were also amplified by RT-PCR and sequenced [Fig. 1].
These clones were designated as HRT1 and HRT2. The
cDNA of HRT1 was 1282 bp long containing an 870 bp
ORF, flanked by a 155 bp 5¢-UTR and a 254 bp 3¢-UTR
including a poly(A) tail of 15 bp (Fig. 1A). The cDNA of
HRT2 was 1051 bp in length containing an 852 bp ORF
(Fig. 1B) and with 92% sequence homolgy to that of
HRT1. The complete cDNA sequences of HRT1 and
HRT2 cloned in this study are available from the DDBJ/
GenBank
TM
/EMBL database under accession numbers
AB061234 and AB064661 respectively. The ORFs of
HRT1 and HRT2 encode 290 and 284 amino acid residues
(87.3% identity) with predicted molecular masses of 33.2
and 32.8 kDa respectively. Hydropathy and transmem-
brane motif analysis of the deduced amino acid sequences
(
TOPPRED
2 program; />interfaces/toppred.html) predicted that both gene products,
HRT1 and HRT2, are hydrophilic proteins having a
putative N-terminus membrane-spanning segment (amino
acid residues 22–42). The deduced amino acid sequences of
HRT1 and HRT2 showed high identities of 31.6, 32.0, 31.6,
29.9 and 30.1% to those of the cis-prenyl chain elongating
enzymes from M. luteus B-P 26 [21], S. cerevisiae (Rer2p
[23], Srt1p [36]), E. coli [22,37] and A. thaliana [24,25]
respectively. Moreover, all of the five highly conserved

regions of cis-prenyl chain elongating enzymes [20–22,34,35]
are found in both the HRT1 and HRT2 sequences [Fig. 2].
These conserved regions are proposed to be important for
the catalytic function, in addition to substrate binding, for
cis-prenyl chain elongating enzymes [33–35,38,39].
Expression analysis of HRT mRNAs
To examine the HRT expression in various Hevea tissues,
Northern blot analysis was carried out by using
32
P-labeled
HRT1 or HRT2 cDNA as the probes. As shown in Fig. 3
(A1 and A2) both probes gave specific hybridization bands
of 1 kb which are consistent with the sizes of each of the
cDNAs. The expression patterns of HRT1 and HRT2
mRNAs among the examined Hevea tissues seem predomi-
nant in latex, whereas little expression was detected in leaves
and shoot tips. However, it is possible that the probes of
HRT1 and HRT2 cross-hybridize with each other because
of the high level of sequence identity. For the study of the
specific expression patterns, RT-PCR analyses were carried
Fig. 2. Comparison of the deduced amino acid sequences of HRT1 and HRT2. The deduced amino acid sequences of HRT1 and HRT2 are compared
with those of cis-prenyltransferases from A. thaliana (GenBank
TM
accession no. AF162441), yeast [Rer2p (Swiss-Prot P35196), Srt1p (Swiss-Prot
Q03175)], M. luteus B-P 26 (GenBank
TM
accession no. AB004319) and E. coli (Swiss-Prot Q47675). The five conserved regions for cis-prenyl chain
elongating enzymes (I to V) are indicated with bars at the top of the sequences. The sequences similar to HRT1 are shaded. Identical amino acid
residues in greater than four of the seven sequences are boxed.
Ó FEBS 2003 cis-Prenyltransferase from Hevea brasiliensis (Eur. J. Biochem. 270) 4675

out by designing specific primers from the low homology
regions of HRT1 and HRT2 coding sequences and each of
the 3¢-UTRs. As shown in Fig. 3B, specific bands of the
RT-PCR products with HRT1 and HRT2 specific primers
could be detected only in the reactions with latex mRNA.
These results indicate the specific expression of HRT1 and
HRT2 in latex, supporting the proposal that the HRT
mRNAs probably function in Hevea latex where natural
rubber is produced.
Overproduction of recombinant HRT proteins
in
E. coli
cells
In order to obtain HRT gene products, the cDNAs were
expressed in E. coli by means of a pET32b(+) expression
system that contains a thioredoxin fusion sequence suitable
for production of a soluble protein in E. coli cytoplasm. By
induction with IPTG the E. coli cells harboring HRT1 or
HRT2 produced recombinant proteins at 52 kDa in the
pellet fraction (Fig. 4, lanes P1 and P2). However, a
detectable amount of HRT2 could be found also in the
soluble fraction of cell-free extract (Fig. 4, lane S2). The
soluble and pellet fractions of HRT2 were subjected to a
metal affinity column for purification of the His-tagged
fusion protein, under native and denatured conditions
respectively. As shown in Fig. 4, the affinity-purified HRT2
protein was the major protein band of 52 kDa (lane P)
corresponding to the His-tagged fusion protein, and when
the fused N-terminal thioredoxin sequence was removed by
enterokinase digestion, a protein of 33 kDa (lane E), was

produced which is attributable to the predicted molecular
mass of the HRT2 protein.
Enzymatic activity of HRT2 proteins
The cell-free homogenates of E. coli BL21(DE3)/pET-
HRT1 and E. coli BL21(DE3)/pETHRT2 were examined
for in vitro rubber transferase activity. Rubber materials
were extracted with a solvent mixture of toluene and hexane
(1 : 1 v/v) after the extraction of medium chain polyprenyl
diphosphates with 1-butanol. The crude homogenate from
HRT2-overexpressing cells showed a slight increase in the
production of polyprenyl diphosphates able to be extracted
with butanol compared to the crude homogenate of the host
cells (Table 1), whereas the HRT1 protein overproduced in
E. coli cells showed no significant increase in enzymatic
activity. Similarly, a slight increase in the radioactivity of the
toluene/hexane extracts was detected after the reaction with
Fig. 3. Expression of HRT mRNAs. (A) Northern blot analysis of
HRT mRNAs. Total RNA samples (15 lg) from: Hevea latex, LT;
leaves, LE; petioles, P; flowers, F; shoot tips, S and roots, R were
separated by agarose gel electrophoresis and transferred onto a nylon
membrane. The blot was hybridized with
32
P-labeled HRT1 or HRT2
cDNA probe. The 0.8–1.4 kb regions of the autoradiogram of the
hybridized membrane, using
32
P-labeled HRT1 (1) or HRT2 (2) as a
probe, are shown. To illustrate equal loading, ethidium bromide
staining of rRNA under UV light is shown (3). (B) RT-PCR analysis of
HRT mRNAs. Two micrograms of total RNA samples from various

Hevea tissues were used as templates. PCR was carried out for
25 cycles using HRT1 or HRT2 specific primers. RT-PCR products
were separated by 1% agarose gel electrophoresis and stained with
ethidium bromide. The 400–600 bp regions of the products, which
were amplified by HRT1 specific primers (1) or HRT2 specific primers
(2), are shown.
Fig. 4. Overexpression of HRT1 and HRT2 in E. coli and purification
of HRT2. The pETHRT1 and pETHRT2 were constructed and
introduced into E. coli BL21(DE3). The expression of these genes were
induced by addition of 0.5 m
M
IPTG. Overexpression of HRT1 and
HRT2 is shown in the left panel: M, molecular mass marker;
SC, supernatant of the homogenate of E. coli BL21(DE3)/pET32b
without IPTG; S1, supernatant of the homogenate of E. coli
BL21(DE3)/pETHRT1 with IPTG; S2, supernatant of the homogen-
ate of E. coli BL21(DE3)/pETHRT2 with IPTG; PC, pellet of the
homogenate of E. coli BL21(DE3)/pET32b without IPTG; P1, pellet
of the homogenate of E. coli BL21(DE3)/pETHRT1 with IPTG; P2,
pellet of the homogenate of E. coli BL21(DE3)/pETHRT2 with IPTG.
Purification of HRT2 is shown in the right panel: M, Molecular mass
marker; P2, pellet of the homogenate of E. coli BL21(DE3)/pET-
HRT2 with IPTG; P, purified HRT2 fusion protein; E, purified HRT2
fusion protein after digestion with enterokinase.
4676 K. Asawatreratanakul et al. (Eur. J. Biochem. 270) Ó FEBS 2003
the crude homogenate of HRT2-overexpressed cells, but not
that of HRT1-overexpressed cells (Table 1).
Because the rubber transferase activities of HRT1 and
HRT2, which were determined by measuring the amount of
rubber material extractable with the toluene/hexane mix-

ture, were not particularly significant, an in vitro rubber
transferase assay was carried out with the addition of fresh
WBP because it has been suggested that these serve as the
site for the initiation of new rubber formation [16,17]. The
amount of [1-
14
C]IPP incorporation in the toluene/hexane
extracts of 2500 dpm that was observed in the control
reaction with WBP is attributable to the endogenous rubber
transferase activity in the WBP. When the homogenates of
BL21(DE3)/pETHRT1 were coincubated with WBP, the
amount of IPP incorporation into the toluene/hexane
extracts was similar to that of the control experiment with
WBP alone. On the other hand, addition of WBP to the
homogenate of BL21(DE3)/pETHRT2 resulted in a
remarkable increase in the amount of IPP incorporation
in the toluene/hexane extracts (Table 1). The increase in
rubber transferase activity over the additive effect of
endogenous activity in WBP clearly indicates that the
rubber transferase activity of HRT2 was enhanced by
various factors in the WBP. The endogenous rubber
transferase activity in the WBP could be denatured most
effectively by heat treatment at 100 °C for 20 min (Table 1).
However, the rubber transferase activity of HRT2 could be
induced partially, even by the addition of boiled WBP,
implying the presence of heat-stable activator(s) in the
WBP, in addition to heat-sensitive ones.
Apparent rubber transferase activity could be obtained
when the purified HRT2 protein was used. Figure 5A
shows the effect of the HRT2 protein on rubber transferase

activity in coincubation with WBP. The activity increased as
the concentration of HRT2 was raised, until the activity
reached saturation. The effect of WBP on the rubber
transferase activity of HRT2, which was assayed in the
presence of 5 lgofHRT2protein,isshowninFig.5B.
The rubber transferase activity increased proportionally
Table 1. In vitro rubber transferase activities in the cell-free homogenates of HRT transformants. The prenyl chain elongating enzyme activity were
assayed in the absence of WBP, in the presence of WBP, and in the presence of boiled WBP as described in Materials and methods. The amounts of
[
14
C]IPP incorporation are averaged from triplicated measurements.
Cell-free homogenate
[
14
C]IPP incorporation (d.p.m.)
BuOH extract Toluene/hexane extract
In the absence of WBP
E. coli BL21(DE3)/pET32b 2670 ± 225 280 ± 68
E. coli BL21(DE3)/pETHRT1 2820 ± 253 280 ± 45
E. coli BL21(DE3)/pETHRT2 3210 ± 244 320 ± 65
In the presence of WBP
WBP 2810 ± 298 2510 ± 218
E. coli BL21(DE3)/pET32b + WBP 3150 ± 173 2230 ± 267
E. coli BL21(DE3)/pETHRT1 + WBP 3700 ± 340 2770 ± 204
E. coli BL21(DE3)/pETHRT2 + WBP 5190 ± 414 7120 ± 356
In the presence of boiled WBP
Boiled WBP 360 ± 62 630 ± 79
E. coli BL21(DE3)/pET32b + boiled WBP 3070 ± 316 980 ± 115
E. coli BL21(DE3)/pETHRT1 + boiled WBP 3440 ± 405 1020 ± 161
E. coli BL21(DE3)/pETHRT2 + boiled WBP 4080 ± 387 3360 ± 292

Fig. 5. Effect of HRT2 and WBP amounts on rubber transferase
activity of HRT2. (A) Effect of HRT2 amount on rubber transferase
activity. In vitro rubber transferase assay was performed in 0.2 mL
reaction volume containing 0.5 mg of WBP and the indicated amount
of purified HRT2 (d)orE. coli BL21(DE3)/pETHRT2 cell-free
homogenate without IPTG (j). After extraction of polyprenyl
diphosphate products with 1-butanol, rubber transferase activity was
measured as described in Materials and methods. (B) Effect of WBP
amount on rubber transferase activity. The rubber transferase was
assayed in the presence of 5 lg of purified HRT2 and the indicated
amount of WBP (d). The control reaction (j) was assayed under
similar conditions without the addition of HRT2.
Ó FEBS 2003 cis-Prenyltransferase from Hevea brasiliensis (Eur. J. Biochem. 270) 4677
to the amount of added WBP and the activity became
saturated when the WBP exceeded  1mg.
Product analysis of the rubber materials in the toluene/
hexane extracts of the HRT2 reactions that were coincu-
bated with WBP, by reversed phase TLC, showed a large
spot of radioactivity at the origin of the TLC plate
indicating a high molecular mass rubber product (data
not shown). Conversely, the reaction of WBP or HRT2
alone gave a small spot at the origin of the TLC plate. In
order to determine the molecular masses of the rubber
products from the reaction of HRT2 coincubated with
WBP, the radioactive products detected at the origin of the
reversed phase TLC were analyzed by GPC (Fig. 6B). The
major radioactivity eluted in the range of molecular mass of
2 · 10
5
)1 · 10

6
Da (corresponding to the rubber polymer
of 3000–15 000 isoprene units), with minor peaks of
radioactivity at 3 · 10
4
,2· 10
3
and 1 · 10
3
Da, equivalent
to  400, 30 and 15 isoprene units respectively, showing
similar distribution of endogenous rubber materials in WBP
(Fig. 6A). When HRT2 was assayed in the absence of WBP,
only a small amount of medium chain polyisoprene
intermediates of the size  2000 to 10
4
Da were produced
(Fig. 6C). The products of the control reaction with WBP
alone (Fig. 6D), show very low quantities of long chain
rubber products whose molecular size distributions are
similar to those in Fig. 6B indicating the low endogenous
rubber transferase activity in WBP. These results suggest
that the long chain rubber molecules (approximate size of
10
5
)10
6
Da) can be produced in larger amounts when the
HRT2 protein is coincubated with WBP.
Functional complementation of yeast mutant stain

SNH23-7D by HRTs
To determine whether HRT1 and HRT2 cDNAs encode
functional enzymes related to the cis-prenyl chain elongating
enzyme in vivo, the cDNAs were expressed in the yeast
mutant strain SNH23-7D, which is deficient in the activity
of dehydrodolichyl diphosphate (dedol-PP) synthase [24].
SNH23-7D shows a temperature-sensitive growth pheno-
type at 37 °C. As shown in Fig. 7, overexpression of HRT2
suppresses the temperature-sensitive growth phenotype of
strain SNH23-7D, whereas the HRT1 showed no effect on
this phenotype of the yeast mutant strain.
Discussion
In this study, we isolated two cDNA clones termed HRT1
and HRT2 that possibly encode the latex cis-prenyltrans-
ferases of the rubber tree H. brasiliensis. Both of the deduced
amino acid sequences, having all of the five highly conserved
regions among cis-prenyl chain elongating enzymes, showed
high homology (87% identity). These regions have been
found to construct the major part of the hydrophobic cleft in
the three-dimensional structure [38], and are important for
the catalytic function as well as the substrate binding of the
enzymes [33–35,39,40]. Therefore, it is reasonable to assign
both HRT1 and HRT2 to a family of cis-prenyl chain elon-
gating enzymes present in the laticifers of H. brasiliensis.
Recently, an A. thaliana gene encoding dedol-PP syn-
thase,wasidentifiedandshowntobehighlyexpressedin
Fig. 6. GPC analysis of the reaction products from HRT2. The reaction
products derived from the in vitro rubber transferase assay were sub-
jected to GPC as described in Materials and methods. (A) Molecular
mass distribution of endogenous rubber materials in WBP, detected by

UV absorption at 210 nm. (B) The molecular mass of
14
C-labeled
products synthesized in vitro by purified HRT2 in the presence of
WBP. (C)
14
C-labeled products synthesized by purified HRT2 alone.
(D)
14
C-labeled products synthesized by WBP alone.
Fig. 7. Functional complementation of the dedol-PP synthase-deficient
yeast strain SNH23-7D by HRT2. Strain SNY9 and SNH23-7D
transformed with plasmid pJR1133, pJRHRT1 and pJRHRT2 were
streaked onto YPD plates and incubated at 23 °Cor37°Cfor4 days.
4678 K. Asawatreratanakul et al. (Eur. J. Biochem. 270) Ó FEBS 2003
roots and leaves [24,25]. On the other hand, in the present
study, Northern blot and RT-PCR analyses of the HRT
genes in various Hevea tissues showed predominant expres-
sion of these genes in the latex. As it has been suggested
previously that rubber biosynthesis takes place only in the
laticifers, however, the specific expression in the latex
strongly suggests a possible functional role of both HRT1
and HRT2 in the Hevea latex as a cis-prenyl chain elongating
enzyme, i.e. rubber transferase.
The majority of HRT1 and HRT2 were expressed as
insoluble fusion proteins in E. coli, which suggests that these
proteins might each be associated with the membrane as a
particle-bound enzyme in a similar manner to rubber
transferase [3,10,40]. Furthermore, hydropathy and trans-
membrane motif analysis revealed that HRT1 and HRT2

sequences have a possible membrane-spanning segment
at amino acid residues 22–42, which is very similar to those
in the dedol-PP synthases cloned from yeast [23] and
A. thaliana [24,25].
Although rubber transferase has been reported previ-
ously, to be bound to rubber particles [13–15], the detailed
mechanism of rubber biosynthesis was not clearly under-
stood. More recently, it has been found that the WBP of
centrifuged fresh Hevea latex is active for in vitro rubber
biosynthesis [16,17], suggesting that the WBP might have
the necessary enzyme systems that are responsible for
rubber biosynthesis. In the current study, we demonstra-
ted that the recombinant HRT2 protein coincubated with
WBP could synthesize medium chain polyprenyl diphos-
phate intermediates as well as long chain rubber, although
HRT2 showed a low cis-prenyl chain elongating enzyme
activity when assayed in the absence of WBP. HRT2 may
possibly function as a cis-prenyl chain elongating enzyme
that cooperates with other activation factors in the WBP
for the biosynthesis of long chain rubber molecules.
Furthermore, the partial enhancement of HRT2 activity
by the addition of the boiled WBP suggests that some
heat-stable compounds in WBP could act as the activator
of rubber transferase. In addition to heat-stable com-
pounds, HRT2 may require other heat-unstable factors in
WBP for complete activation.
Suppression of HRT2 on the temperature-sensitive
growth of yeast mutant strain SNH23-7D shows clearly that
HRT2 functions as a cis-prenyltransferase and produces
dedol-PP, whose prenyl chain length is compatible with that

of yeast dolichols (C
80
–C
100
; 1.1–1.3 kDa). GPC analysis of
the rubber material synthesized by the action of purified
HRT2 in the absence of WBP indicated the production of a
small amount of medium chain polyisoprene intermediates
of molecular size between 10
3
and 10
4
Da, which are smaller
than those produced by the coincubation of HRT2 with
WBP (2 · 10
5
)1 · 10
6
Da) but similar to the molecular size
of dolichols in yeast (Fig. 6C). Furthermore, HRT2 showed
a little activity for the production of prenyl diphosphates
extractable with butanol. Taken altogether, two probable
mechanisms of natural rubber biosynthesis including HRT2
as well as the other factor(s) in WBP could be deduced as
follows (a) HRT2 synthesizes medium chain polyprenyl
diphosphate whose chain lengths are comparable to those of
dolichols ( 10
3
Da), then various factors in WBP combine
the medium chain polyprenyl products to synthesize high

molecular mass rubber materials, and (b) HRT2 catalyzes
further prenyl chain elongation to synthesize linear high
molecular mass polyprenyl products with the cooperation of
various factors in WBP that remarkably enhance HRT2
activity.
We were not able to detect the rubber transferase activity
in any fraction of HRT1 protein overproduced in E. coli.
These results led us to speculate that the 35 amino acids
mismatch between HRT1 and HRT2 may be effective on
their native structures and also important for their enzy-
matic functions. The high similarity between HRT1 and
HRT2 suggests the possibility that HRT1 may encode a cis-
prenyl chain elongating enzyme such as dedol-PP synthase
whose function in the rubber tree is still unknown.
Tateyama et al. analyzed the polyisoprenoid alcohols of
H. brasiliensis using a two-plate TLC method [41], and
established that polyprenols and dolichols are found with
prenyl chain-length distribution of around C
50
–C
105
in
several tissues of H. brasiliensis. In addition, we found many
protein sequences that originated from H. brasiliensis in
protein databases, showing high homology to cis-prenyl-
transferase, such as AAM92880 (AAM92889, AAM92890),
AAM92881, AAM92879, BAB92023 (AAM92883,
AAM92884, AAM92885, AAM92887, AAM92888),
BAB92024 and AAM92882 (AAM92886) (submitted to
Genbank

TM
and DDBJ by Coldren et al. and Sando et al.
respectively). However, most of these amino acid sequences,
except for that of AAM92882, are similar to that of HRT1
(more than 98% identity) but not to HRT2. None of them
have been proved to show rubber transferase activity.
The precise mechanisms of rubber biosynthesis in
H. brasiliensis are not yet well understood. The results of
our study of Hevea rubber transferase represent an import-
ant step in understanding the process of rubber biosynthesis
in rubber-producing plants at the molecular level and
provide a basis for further investigation of the molecular
mechanism of HRT reaction. More biochemical studies
with the purified HRT are required to further characterize
the involvement of this enzyme in rubber biosynthesis,
especially on the molecular analysis of the activator(s) in
WBP.
Acknowledgements
We are grateful to Dr A. Nakano and Dr M. Sato (RIKEN, Japan) for
kindly providing the yeast strains SNH23-7D and SNY9, and Dr A.
Ferrer (University of Barcelona, Spain) for kindly providing the yeast
expression vector pJR1133. This work was supported in part by
Grants-in-Aid for Scientific Research (12480169 to T.K. and 13680667
to Y W.Z.) from the Ministry of Education, Science and Culture of
Japan, and by the Asahi Glass-, Heiwa–Nakajima-, Sumitomo- and
Goho-Life Science Foundations.
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