(Received for publication, May 30, 1995)
From the
The genes encoding two dissociable components essential for Bacillus stearothermophilus heptaprenyl diphosphate synthase (all-trans-hexaprenyl-diphosphate:isopentenyl-diphosphate
hexaprenyl-trans-transferase, EC 2.5.1.30) were cloned, and
their nucleotide sequences were determined. Sequence analyses revealed
the presence of three open reading frames within 2,350 base pairs,
designated as ORF-1, ORF-2, and ORF-3 in order of nucleotide sequence,
which encode proteins of 220, 234, and 323 amino acids, respectively.
Deletion experiments have shown that expression of the enzymatic
activity requires the presence of ORF-1 and ORF-3, but ORF-2 is not
essential. As a result, this enzyme was proved genetically to consist
of two different protein components with molecular masses of 25 kDa
(Component I) and 36 kDa (Component II), encoded by two of the three
tandem genes. The protein encoded by ORF-1 has no similarity to any
protein so far registered. However, the protein encoded by ORF-3 shows
a 32% similarity to the farnesyl diphosphate synthase of the same
bacterium and has seven highly conserved regions that have been shown
typical in prenyltransferases (Koyama, T., Obata, S., Osabe, M.,
Takeshita, A., Yokoyama, K., Uchida, M., Nishino, T., and Ogura, K.
(1993) J. Biochem. (Tokyo)113,
355-363).
Prenyltransferases catalyze the fundamental isoprenoid chain
elongation to produce prenyl diphosphates with various chain lengths
and stereochemistries, which are led to such diverse isoprenoid
compounds as steroids, carotenoids, glycosyl carrier lipids, prenyl
quinones, and prenyl proteins(1, 2) . These enzymes
can be classified into four groups according to the mode of requirement
for enzymatic activity (Table 1). Short-chain prenyl diphosphate
synthases such as farnesyl-(3) , and geranylgeranyl- (4) diphosphate synthases require no cofactor except divalent
metal ions such as Mg
On the other hand, hexaprenyl
(C Very few precedents of such enzymes have so far
been reported. Coenzyme B In order to shed light on the significance of such an unusual
two-component system for prenyl diphosphate synthesis and the role of
each component in functional expression, we cloned and sequenced the
genes encoding the HepPP synthase of Bacillus
stearothermophilus. This is the first report of the genes encoding
two essential proteins involved in prenyl diphosphate synthesis.
As the Southern blot analysis
with the KpnI-HindIII fragment of pCR64 gave a 3-kbp
band in the hybridization pattern of the genomic DNA digested with AccI (data not shown), a genomic library of B.
stearothermophilus was constructed with approximately 3-kbp
fragments and inserted into the SmaI site of pUC18. E.
coli JM109 transformants (>6,000 colonies) were screened by
colony hybridization using digoxigenin (DIG, Boehringer
Mannheim)-labeled pCR64 fragment as a probe. A clone, pAC2, containing
a 2.5-kbp insert, was isolated and sequenced. There were three open
reading frames in the same strand of pAC2, and they were designated as
ORF-1, ORF-2, and ORF-3 in order of the nucleotide sequence, which
encoded proteins of 220, 234, and 323 amino acids, respectively. The
protein encoded by ORF-3 contains seven conserved regions, including
the two aspartate-rich sequences,
LXXDDXXDXXRRG and
GXXFQXXDDXXD, which are typical of
prenyltransferases(20, 23) . In order to specify
the enzyme that is encoded by ORF-3, we transformed, with pAC2, E.
coli cells that do not produce HepPP synthase but produce
octaprenyl (C In order to add an upstream portion to the 2.5-kbp insert in pAC2,
another PCR with an M13 primer and an antisense primer of pAC2 (from
position 2098 to 2079) was carried out using the genomic library as the
template. A 2-kbp clone, pPR2 was obtained and its BamHI
fragment was replaced with the corresponding sequence (from position
1975 to 3088). The resulting clone, designated as pTL6, was found to be
longer by 423 bp than pAC2 (Fig. 1). After transformation, E. coli JM109 cells harboring pTL6 were homogenized and then
heat-treated at 55 °C for 60 min. Incubation with the cell-free
homogenate gave polyprenyl products extractable with butanol, which
were then converted to the corresponding alcohols by acid phosphatase
treatment(21) . As shown in Fig. 2, it is clear that the
cells harboring pTL6 express thermostable HepPP synthase, producing
HepPP as well as some intermediate shorter-chain prenyl diphosphates.
Figure 1:
Schematic diagram of the clones
obtained by hybridizations or deletions. Arrows indicate the
three open reading frames ORF-1, ORF-2, and ORF-3. Restriction sites
are abbreviated as follows: B, BamHI; E, EcoT14I; Hi, HincII; Sa, SacI; Sc, ScaI. Numbers indicate
the positions of the nucleotide ends based on those of
pTL6.
Figure 2:
TLC
radiochromatogram of the alcohols obtained by enzymatic hydrolysis of
the products formed by the incubation with the cell-free homogenate of E. coli JM109/pTL6. The products were analyzed as described
under ``Experimental Procedures.'' Arrowheads indicate the positions of authentic alcohols: C
Although pAC2 contains the three open reading frames, the E.
coli cells transformed with pAC2 did not express HepPP synthase
activity. However, when an out-of-frame clone with lacZ region
in the pUC18 vector was prepared, a slight HepPP synthase activity was
detected, indicating that a fusion protein of a lacZ-ORF-1
product might be produced by pAC2 (data not shown).
Comparison of the deduced amino
acid sequences of the proteins encoded by the three open reading frames
with those for FPP synthases from B.
stearothermophilus(20) , E. coli(24) ,
and Saccharomyces cerevisiae(25) , for GGPP synthase
from Sulfolobus acidocaldarius(26) , and for HexPP
synthase from S. cerevisiae(27) indicated that only
the ORF-3 protein has a significant level of similarity to the above
mentioned prenyltransferases. As shown in Fig. 3, the ORF-3
protein has seven highly conserved regions that are typical of prenyl
diphosphate synthases(20) . This protein shows 31.9% similarity
to that of the FPP synthase of the same bacterium. The protein deduced
from ORF-2 shows 39.1% similarity to an unknown protein deduced from
the o-251 gene of E. coli(28) .
Figure 3:
Comparison of amino acid sequences of the
ORF-3 encoding protein and several prenyltransferases. FPP synthases: 1, from B. stearothermophilus; 2, from E. coli; 3, from S. cerevisiae; 4,
GGPP synthase from S. acidocaldarius; 5, HexPP
synthase from S. cerevisiae; 6, ORF-3 protein. Amino
acids identical for at least 4 out of 6 sequences in the conserved
regions (I-VII) are shaded.
HepPP synthase is one of the three prenyl diphosphate
synthases in B. stearothermophilus. It produces the precursor
of the respiratory quinone side chain in this bacterium(29) .
The equivalent from B. subtilis and the HexPP synthase from M. luteus B-P 26 (9, 10, 11) are
unique in that each of them comprises two dissociable protein
components, neither of which shows any enzyme
activity(9, 13) . By taking advantage of the
thermostability of B. stearothermophilus enzyme and the lack
of HepPP synthase in E. coli, we succeeded in the first
identification of the genes encoding such two components that
constitute a medium-chain polyprenyl diphosphate synthase. On the basis
of the data from the deletion experiments, we concluded that both ORF-1
and ORF-3 are essential for the HepPP synthase activity. It is very
interesting why this enzyme must take such a two-dissociable component
system in contrast to most of the other prenyl diphosphate synthases
that are tightly coupled homodimers. In the case of protein
farnesyltransferase and protein geranylgeranyltransferase, these
transferases are heterodimers that share a common In experiments with a yeast mutant
in coenzyme Q biosynthesis, Ashby and Edwards (27) have
isolated a gene from a plasmid containing a wild-type genomic DNA
fragment that is able to complement the mutant and restore HexPP
synthase activity. They have also shown that this gene encodes a
473-amino acid protein having highly conserved domains characteristic
of prenyl diphosphate synthases as shown in Fig. 3. Therefore,
this protein seems to correspond to the product of ORF-3 in this
report. However, it is not known whether the yeast 473 amino-acid
protein acts as HexPP synthase by itself or in association with another
gene product similar to the ORF-1 protein. If the latter is the case,
the mutant described above must be deficient in one of the two
components of HexPP synthase. Similar to this is the case of the yeast
mutant dpr1/ram1, which lacks protein: farnesyltransferase
activity(32) . The wild-type DPR1/RAM1 gene product
expressed in E. coli is catalytically inactive but becomes
active when it is mixed with an extract of the dpr1/ram1 cells, suggesting that DPR1/RAM1 encodes one of the two
subunits of this transferase. It is interesting to learn whether
dissociable heterodimeric systems are common to the medium-chain prenyl
diphosphate synthases of both prokaryotic and eukaryotic cells.
Overproduction and purification of each component are in progress for
further exploring the significance and mechanistic enzymology of this
unusual two-component system.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s) D49975 [GenBank]and D49976[GenBank].
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, Zn
, or
Mn
, which are commonly required by all
prenyltransferases. The enzymes that catalyze the formation of (Z)-polyprenyl chains including undecaprenyl-(5) ,
nonaprenyl-(6) , and dehydrodolichyl- (7) diphosphate
synthases require phospholipid or detergent. The enzymes catalyzing the
synthesis of long-chain (E)-prenyl diphosphates, including
octaprenyl- (C
), solanesyl- (C
, all-E-nonaprenyl-) and decaprenyl- (C
)
diphosphates, require protein factors that remove polyprenyl products
from their active sites to facilitate and maintain the turnover and
catalysis(8) .
) diphosphate (HexPP) (
)synthase from Micrococcus luteus B-P 26 (9, 10, 11) and heptaprenyl (C
)
diphosphate (HepPP) synthase from Bacillus
subtilis(12, 13) , which catalyze the synthesis
of medium-chain (E)-prenyl diphosphates, are unusual because
they do not require lipid or detergent but comprise two non-identical
protein components. These components exist without binding with each
other under physiological conditions, and neither of them has any
catalytic activity.
-dependent diol dehydratase (EC
4.2.1.28) consists of two different components, F and S(14) ,
which are easily separable in the absence of its substrate. Rab
geranylgeranyltransferase from rat brain is a heterodimer, and it
separates into two subunits in the presence of high salt concentrations (15) . Ras farnesyltransferase also consists of two
nonidentical subunits, but these subunits can be separated from each
other only after denaturation with urea or guanidine(16) .
Materials
[1-C]Isopentenyl
diphosphate (IPP) (1.95 GBq/mol) was a product of Amersham Corp.
Nonlabeled IPP and (E,E)-farnesyl diphosphate (FPP) were
synthesized according to the procedure of Davisson et
al.(17) . Lysozyme, deoxyribonuclease, and acid
phosphatase were purchased from Boehringer Mannheim. Precoated reversed
phase thin layer chromatography (TLC) plates (LKC-18) were products of
Whatman. T4 DNA ligase and DNA polymerase were purchased from Takara
Shuzo Co., Ltd., Japan. B. stearothermophilus ATCC 10149 was
obtained from American Type Culture Collection. All other chemicals
were of analytical grade.
General Procedures
Restriction enzyme digestion,
transformation, and other standard molecular biology techniques were
carried out as described by Sambrook et al.(18) .Genomic DNA Preparation
B. stearothermophilus grown in 1 liter of L-B medium (18) at 55 °C was
harvested in the late log phase, and the chromosomal DNA was isolated
as described by Saito and Miura(19) . The genomic DNA was
partially digested with Sau3AI, and the resulting fragments
were used as polymerase chain reaction (PCR) templates.PCR Cloning of a Region of HepPP Synthase
Gene
Nine degenerate oligonucleotide primers were designed from
the amino acid sequences of highly conserved regions typical of
prenyltransferases (20) : sense primers, p9 (region I) ()5`-YTNGARGCNGGNGGNAAR(CA)G-3`; p1 (region II)
5`-CTNAT(ACT)CAYGAYGAYYTNCCNTCNATGGAC-3`; p2 (region II)
5`-GAYAAYGAYGAYYTN(CA)GN(CA)GNGGC-3`; p10 (region II) 5`-TAY(TA)
(CG)NYTNAT(TCA)CAYGAYGA-3`; antisense primers, p11 (region IV)
5`-YTCCATRTCNGCNGCYTGNCC-3`; p4 (region VI)
5`-ATCRTCNC(TG)DATYTGRAANGCNARNCC-3`; p6 (region VI)
5`-ATCNARDATRTCRTCNC(TG)DATYTGRAA-3`; p8 (region VI)
5`-GTCRCTNCCNACNGGYTTNCC-3`; p13 (region VI) 5`-DATRTCNARDATRTCRTC-3`,
where R is A or G, Y is C or T, D is G, A, or T, and N is A, G, C, or
T. PCR was performed in a final volume of 100 µl containing 10
mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl
, 0.001% (w/v) gelatin, 200 µM each
dNTPs, 100 pmol of amplification primer pairs (p1/p4, p1/p6, p1/p8,
p2/p4, p2/p6, p2/p8, p9/p11, p9/p4, p9/p6, p9/p8, p9/p13, p1/p11,
p2/p11, p1/p13, p2/p13, p10/p4, p10/p6, p10/p8, or p10/p13), 1 unit of
DNA Polymerase Enhancer (Stratagene), 500 ng of genomic DNA fragments
digested with Sau3AI, and 2 units of Taq polymerase.
The protocol used was 35 cycles of PCR of 30 s at 94 °C, 30 s at 50
°C, and 1 min at 72 °C, followed by extension at 72 °C for
7 min (Perkin-Elmer TCI thermal cycler). Samples of reaction mixtures
were subjected to electrophoresis on a 1.5% agarose gel. All DNA
products of PCR were purified, subcloned into a pT7Blue T-Vector, and
sequenced by the dideoxy chain termination method.
Southern Blot Analysis
Genomic DNA was digested
with various restriction enzymes. Electrophoresis, blotting, and
hybridization were performed according to the standard
methods(18) .Construction of B. stearothermophilus Genomic
Library
The restriction fragments of B. stearothermophilus genomic DNA were fractionated with 0.8% agarose electrophoresis.
Approximately 3 kbp of AccI fragments were isolated by
electrophoretic elution into a dialysis tube. The AccI
fragments were treated with T4 DNA polymerase, and the resulting
fragments were ligated to the SmaI site of pUC18, after which Escherichia coli JM109 cells were transformed with the
resulting plasmids.Cloning of the Prenyltransferase
Gene
Approximately 6,000 transformant colonies were screened by
colony lift hybridization for the presence of plasmids bearing the
genomic fragment hybridizing to the probe. Candidate colonies that
hybridized to the probe were picked from the master plate and plasmid
DNA was prepared.Preparation of Cell-free Homogenate of E. coli
Transformants
The transformed E. coli cells were
cultured in 30 ml of L-B medium at 37 °C overnight. The cells were
harvested by centrifugation and then homogenized by sonication
according to our procedure for the screening of thermostable prenyl
diphosphate synthase activity(20) .HepPP Synthase Assay
The assay mixture contained,
in a final volume of 1.0 ml, 50 mM Tris-HCl buffer (pH 8.5),
25 mM MgCl, 50 mM NH
Cl, 50
mM 2-mercaptoethanol, 25 µM FPP, 0.46 µM [1-
C]IPP (1.95 GBq/mol), and 500 µl of
the crude cell-free homogenate to be examined. The incubation was
carried out at 55 °C for 3 h, and then the reaction mixture was
treated with 1-butanol to extract the product of the prenyltransferase
reaction. The radioactivity in the butanol extract was measured with an
Aloka LSC-1000 liquid scintillation counter.
Product Analysis of the Reaction Catalyzed by
Prenyltransferase Expressed in E. coli
After the enzymatic
reaction at 55 °C, the radioactive prenyl diphosphate in the
reaction mixture was hydrolyzed to the corresponding alcohol with
potato acid phosphate according to our method reported
previously(21) . The alcohol was extracted with pentane and
analyzed by TLC on reversed phase LKC-18 in a solvent system of
acetone/water (19:1). The positions of authentic standards were
visualized with iodine vapor, and the distribution of radioactivity was
determined by autoradiography. The TLC plates were exposed on a Fuji
imaging plate at room temperature for 1 day. The exposed imaging plate
was analyzed with a Fuji BAS 2000 bioimage analyzer.DNA Sequence Analysis
The nucleotide sequences was
determined by the dideoxy chain termination method with a DNA sequencer
(Applied Biosystems, model 373A). Computer analysis and comparison of
DNA sequence were performed using GENETYX genetic information
processing software (Software Development).Deletion of pTL6
A HindIII site was
introduced at position 220 of pTL6 by site-directed mutagenesis using a
mutagenic oligonucleotide, 5`-GGGAAAAAGTAAGCTTGCAAATGTCTAGC-3`
according to the procedure described in our previous
paper(22) . After deletion of the HindIII fragment
(220 bp) of the resulting plasmid, the residual DNA fragment was
ligated to give a plasmid designated as pTLM17. After deletion of the SacI (1,027 bp) or the HincII (1,032 bp) fragment
from pTL6, the residual DNA fragment was ligated, and the resulting
plasmid was designated as pTLD7 or pTLD9, respectively. After deletion
of the EcoT14I-ScaI (368 bp) fragment of pTLM17, the
residual DNA was treated with T4 DNA polymerase and ligated, and the
resulting plasmid was designated as pTLD17, After transformation of E. coli cells with these plasmids, the cells harboring
deletion plasmids were examined for production of the thermostable
HepPP synthase.
Isolation of the Gene for HepPP Synthase
The
genomic DNA of B. stearothermophilus was chosen for this
purpose, because this bacterium contains heat-stable prenyltransferases
including HepPP synthase in addition to FPP synthase, whose gene has
already been cloned and characterized(20) . In order to obtain
possible probes that hybridize genes for prenyltransferases including
HepPP synthase, we synthesized nine degenerate oligonucleotide primers
of 18-30 bp long designed on the basis of conserved amino acid
regions of prenyltransferases. After 19 PCRs using partially digested
genomic DNAs of B. stearothermophilus as templates, we
obtained 37 clones. These PCR products were cloned into a pT7Blue
T-Vector, and their nucleotide sequences were determined. Ten clones
were found to have the same sequences as fragments of fps gene(20) , but we found that three similar clones of
approximately 500 bp had the same sequence encoding a typical
prenyltransferase motif, DDXXD. One of the plasmids, pCR64 was
used as a probe for subsequent screening of the clones containing the
entire coding region of a prenyltransferase other than the FPP synthase
of B. stearothermophilus.) diphosphate synthase instead. After
denaturation of the intrinsic prenyltransferases derived from the host
cells by heat treatment, the cell-free homogenate was assayed for
heat-stable prenyltransferase. However, no heat-stable
prenyltransferase activity was detected in the cell-free homogenate.
, (all-E)-farnesol; C
, (all-E)-geranylgeraniol; C
, (all-E)-farnesylgeraniol; C
, (all-E)-heptaprenol; C
,
(2Z,6Z,10Z,14Z,18Z,22Z,26Z,30Z,34E,38E)-undecaprenol; C
,
(2Z,6Z,10Z,14Z,18Z,22Z,26Z,30Z,34Z,38E,42E)-dodecaprenol; orig., origin; S. F., solvent
front.
Characterization of HepPP Synthase Genes
The
nucleotide sequence of the 3-kbp insert of pTL6 contains three open
reading frames, ORF-1, ORF-2, and ORF-3, which are the same
constituents of pAC2. ORF-1 begins with an ATG codon at position 451
and terminates with the TAG codon at position 1110, encoding a
220-amino acid protein with a calculated molecular weight (M) of 24,610. ORF-2 begins with the ATG codon at
position 1118 and contains 702 nucleotides encoding a 234-amino acid
protein with an M
of 27,132. ORF-3, beginning with
the GTG codon at position 1829, encodes a 323-amino acid protein with
an M
of 36,172.
Identification of HepPP Synthase Gene
In order to
specify the real structural genes responsible for the HepPP synthase
activity, we prepared several plasmids having DNA inserts with some
deletions in pTL6 (Fig. 1) and examined the enzymatic activity
in each transformant. As shown in Table 2, neither pTLD7, which
had deletion from position 2062 to the end, nor pTLD9, which lacked 582
bp in ORF-1, expressed any thermostable prenyltransferase activity.
However, the plasmid pTLD17, from which 367 bp in ORF-2 was lost,
showed a significant level of HepPP synthase activity. These results
indicate that ORF-1, as well as ORF-3, is essential for the HepPP
synthase activity whereas ORF-2 is not essential.
subunit in
association with different
subunits(30) . It has also
been suggested that
- and
subunits bind a prenyl diphosphate
and a protein substrate, respectively(16) . This may raise the
possibility that the two components of HepPP synthase are responsible
for the respective bindings for the prenyl donor (allylic diphosphate)
and for the acceptor (IPP). However, this possibility seems unlikely
for the following reasons. Comparison of the deduced amino acid
sequence of ORF-3 with those of other prenyl diphosphate synthases
revealed that it has conserved regions that involve the two putative
binding sites for the allylic and homoallylic substrates(31) .
In contrast, the protein encoded by ORF-1 has no such similarity, nor
did we find similar protein entries in protein data bases. Therefore,
it seems likely that the protein encoded by ORF-3 carries substantial
sites for substrate binding and catalysis, whereas the protein by ORF-1
plays an auxiliary but essential role in catalytic function. This would
be consistent with our previous observations that one of the two
components of M. luteus HexPP synthase or B. subtilis HepPP synthase is more heat-stable than the other. We have also
demonstrated that the heat-stable components of these enzymes are so
specific for their own partners that one cannot substitute for the
other(13) . This is in contrast to the case of long-chain
prenyl diphosphate synthases, which are stimulated by commonly
effective protein factors(26) . The medium-chain- (C
and C
) and long-chain- (C
,
C
, and C
) prenyl diphosphate synthases
catalyze quite similar reactions, sharing the starting substrates. The
only difference between these two classes of enzyme lies in the chain
length of the product. Probably, the C
and C
products are amphipathic, whereas the C
and longer
products are too hydrophobic to form micelles in an aqueous phase.
Taken together, these observations suggest that the unique
constitutions of the medium-chain prenyl diphosphate synthases are
related to their abilities to catalyze the synthesis of amphipathic
products from soluble substrates.
We are grateful to Takuya Toyokawa and Zhang Yuanwei
for cooperation in the analysis of the enzymatic activities.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.