©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Nucleotide Sequences of the Genes for Two Essential Proteins Constituting a Novel Enzyme System for Heptaprenyl Diphosphate Synthesis (*)

(Received for publication, May 30, 1995)

Ayumi Koike-Takeshita (1) Tanetoshi Koyama (2)(§)(¶) Shusei Obata (1) Kyozo Ogura (2)(¶)

From the  (1)Bio Research Laboratory, Toyota Motor Corporation, Toyota-cho 1, Toyota, Aichi 471-71 and the (2)Institute for Chemical Reaction Science, Tohoku University, Aoba-ku, Sendai, Miyagi 980, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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).


INTRODUCTION

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, 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) .



On the other hand, hexaprenyl (C) diphosphate (HexPP) (^1)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.

Very few precedents of such enzymes have so far been reported. Coenzyme B-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) .

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.


EXPERIMENTAL PROCEDURES

Materials

[1-^14C]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) (^2)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(2), 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(2), 50 mM NH(4)Cl, 50 mM 2-mercaptoethanol, 25 µM FPP, 0.46 µM [1-^14C]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.


RESULTS

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.

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) 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.

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, (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.



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).

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(r)) 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(r) of 27,132. ORF-3, beginning with the GTG codon at position 1829, encodes a 323-amino acid protein with an M(r) of 36,172.

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.



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.




DISCUSSION

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 alpha subunit in association with different beta subunits(30) . It has also been suggested that alpha- and beta 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.

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.


FOOTNOTES

*
This work was supported by Grant-in-aid for Scientific Research on Priority Areas 06240102 from the Ministry of Education, Science and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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].

§
Present address: Dept. of Biochemistry and Engineering, Faculty of Engineering, Tohoku University, Aoba-ku, Sendai 980-77, Japan.

To whom correspondence should be addressed. Tel.: 81-22-217-5621; Fax: 81-22-217-5620; ogura{at}icrs.tohoku.ac.jp.

^1
The abbreviations used are: HexPP, hexaprenyl diphosphate; HepPP, heptaprenyl diphosphate; IPP, isopentenyl diphosphate; FPP, (E,E)-farnesyl diphosphate; GPP, geranyl diphosphate; GGPP, geranylgeranyl diphosphate; TLC, thin layer chromatography; PCR, polymerase chain reaction; bp, base pair(s); kbp, kilobase pair(s); ORF, open reading frame.

^2
Regions I-VII correspond to regions A-G in our previous report (20).


ACKNOWLEDGEMENTS

We are grateful to Takuya Toyokawa and Zhang Yuanwei for cooperation in the analysis of the enzymatic activities.


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