Identification of a Novel Gene Cluster Participating in Menaquinone (Vitamin K2) Biosynthesis
CLONING AND SEQUENCE DETERMINATION OF THE 2-HEPTAPRENYL-1,4-NAPHTHOQUINONE METHYLTRANSFERASE GENE OF BACILLUS STEAROTHERMOPHILUS*

(Received for publication, February 10, 1997)

Ayumi Koike-Takeshita Dagger , Tanetoshi Koyama § and Kyozo Ogura par

From the Dagger  Bio Research Laboratory, Toyota Motor Corporation, Toyota-cho 1, Toyota, Aichi 471-71, the § Department of Biochemistry and Engineering, Faculty of Engineering, Tohoku University, Aoba-ku, Sendai 980-77, and the par  Institute for Chemical Reaction Science, Tohoku University, Aoba-ku, Sendai, Miyagi 980-77, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

We recently described the isolation and sequence analysis of a DNA region containing the genes of Bacillus stearothermophilus heptaprenyl diphosphate synthase, which catalyzes the synthesis of the prenyl side chain of menaquinone-7 of this bacterium. Sequence analyses revealed the presence of three open reading frames (ORFs), designated as ORF-1, ORF-2, and ORF-3, and the structural genes of the heptaprenyl diphosphate synthase were proved to consist of ORF-1 (heps-1) and ORF-3 (heps-2) (Koike-Takeshita, A., Koyama, T., Obata, S., and Ogura, K. (1995) J. Biol. Chem. 270, 18396-18400). The predicted amino acid sequence of ORF-2 (234 amino acids) contains a methyltransferase consensus sequence and shows a 22% identity with UbiG of Escherichia coli, which catalyzes S-adenosyl-L-methionine-dependent methylation of 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone. These pieces of information led us to identify the ORF-2 gene product. The cell-free homogenate of the transformant of E. coli with an expression vector of ORF-2 catalyzed the incorporation of S-adenosyl-L-methionine into menaquinone-8, indicating that ORF-2 encodes 2-heptaprenyl-1,4-naphthoquinone methyltransferase, which participates in the terminal step of the menaquinone biosynthesis. Thus it is concluded that the ORF-1, ORF-2, and ORF-3 genes, designated heps-1, menG, and heps-2, respectively, form another cluster involved in menaquinone biosynthesis in addition to the cluster of menB, menC, menD, and menE already identified in the Bacillus subtilis and E. coli chromosomes.


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.

Heptaprenyl diphosphate (HepPP)1 synthase is one of the three prenyl diphosphate synthases in Bacillus stearothermophilus. It catalyzes the condensations of four molecules of isopentenyl diphosphate (IPP) with farnesyl diphosphate (FPP) to give HepPP, the precursor of the menaquinone (MK) side chain in this bacterium.

MK, or 2-methyl-3-prenyl-1,4-naphthoquinone, is a lipophilic nonprotein component of the electron transport chain in bacteria. In addition, MK is necessary for successful endospore formation and is involved in regulation of cytochrome formation in Bacillus subtilis (1). MK is synthesized in bacteria from isochorismate through a series of MK-specific reactions (Fig. 1). Five MK biosynthetic genes, menA, menB, menC, menD, and menE, have been identified, and four of them are shown to be clustered on the chromosomes of Escherichia coli (2-5) and B. subtilis (6, 7), although menC has not yet been identified in B. subtilis (8).


Fig. 1. Biosynthetic pathway of menaquinone. The abbreviations used are as follows: TPP, thiamine pyrophosphate; SHCHC, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate; OSB, o-succinyl benzoate; OSB-CoA, o-succinyl benzoate-coenzyme A; DHNA, 1,4-dihydroxy-2-naphthoic acid; DMK, demethyl-MK. Italics show the genetic loci in mutant strains of each separate reaction step.
[View Larger Version of this Image (6K GIF file)]

Previously, we reported the cloning of the genes that encode the HepPP synthase of B. stearothermophilus (9), which is composed of two nonidentical protein components. Sequence analyses of the gene regions revealed the presence of three open reading frames designated as ORF-1, ORF-2, and ORF-3. After several deletion experiments, we concluded that the structural genes of the HepPP synthase were ORF-1 and ORF-3, designated heps-1 and heps-2, respectively. Then the next question is what is the function of ORF-2, which is located between the two structural genes of HepPP synthase.

A data base search for proteins similar to the predicted product of ORF-2 (234 amino acids) indicated that the ORF-2 product contains the methyltransferase consensus sequence and shows a 69% identity with GerC2, whose gene is one of the genes in the gerC cluster of B. subtilis, which is involved in spore germination (10), a 22% identity with UbiG of E. coli, which catalyzes S-adenosyl-L-methionine-dependent methylation of 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone (11), and a 37% identity with the O251 of E. coli, whose function is not yet known (12).

To identify the function of ORF-2 as the 2-heptaprenyl-1,4-naphthoquinone methyltransferase gene, we constructed an expression system of ORF-2 and examined the enzymatic activity for catalysis of the terminal step in MK biosynthesis. This is the first report on the cloning of the gene encoding the methyltransferase catalyzing the terminal step in MK biosynthesis as well as on the existence of another gene cluster involved in MK biosynthesis.

An E. coli mutant, AN70, is deficient of a specific methylase of the ubiquinone biosynthetic pathway and is completely devoid of MK and contains high levels of demethylmenaquinone (22). We also examined complementation of the growth of AN70 in M9 medium by the expression vector of o251.


EXPERIMENTAL PROCEDURES

Materials

[1-14C]Isopentenyl diphosphate (IPP; 2.22 GBq/mmol) and S-adenosyl-L-[methyl-14C]methionine (14C-SAM; 2.29 GBq/mmol) were products of Amersham Corp. Nonlabeled IPP and FPP were synthesized according to the procedure of Davisson et al. (13). Nonlabeled SAM was purchased from Sigma. Precoated reversed phase TLC plates (LKC-18) were products of Whatman. T4 DNA ligase and DNA polymerase were purchased from Takara Shuzo Co., Ltd. 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. (14).

Bacterial Strains and Media

E. coli K12 strain JM109 was used as the host to express the recombinant proteins. E. coli strains containing plasmid vectors conferring ampicillin resistance were maintained on Luria-Bertani medium supplemented with 50 µg/ml ampicillin. An E. coli strain AN70 used for complementation experiments was kindly supplied by Drs. G. Unden and I. Z. Young.

Construction of Expression Vector

The original cloning of the B. stearothermophilus HepPP synthase gene cluster, ORF-1 (heps-1), ORF-2, and ORF-3 (heps-2) in pT7 Blue-T vector as pTL6 was previously described (9). To introduce a SphI site at 5' end and a HindIII site at 3' end of ORF-2, mutagenic oligonucleotide primers, 5'-AAGGGTAGAAGCATGCGTCAATCG-3' (mismatched bases are underlined) and 5'-CATCGCCTTAAGCTTCATGTTGTTCACC-3' were synthesized, respectively. Conditions for polymerase chain reaction (PCR) were: 35 cycles of denaturation at 94°C for 30 s, annealing at 65°C for 30 s, and extension at 72°C for 1 min, followed by extension at 72°C for 7 min. To a final volume of 100 µl, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% (w/v) gelatin, 200 µM each of dNTPs, 100 pmol of the amplification primer pair, 1 unit of DNA polymerase enhancer (Stratagene), approximately 1 ng of pTL6, and 2 units of Taq polymerase were added. The PCR products were digested with SphI, treated with T4 DNA polymerase, and digested again with HindIII. The samples were subjected to electrophoresis on a 0.8% agarose gel. Approximately 0.7-kilobase pair fragments were isolated, cloned into pTrc99A vector (Pharmacia Biotech Inc.), which was digested with NcoI, treated with T4 DNA polymerase, and digested with HindIII. The resulting plasmid was designated pHE84.

To introduce an NcoI site at 5' end and a HindIII site at 3' end of o251 of E. coli, mutagenic oligonucleotide primers, 5'-GAGCAGGCATTGCCATGGTGG-3' (mismatched bases are underlined) and 5'-AAAAAGCTTTTCCGGTCTCC-3' were synthesized. PCR was carried out under the same conditions as described above, using the genomic DNA from E. coli as the template. Then the PCR products were subjected to electrophoresis on a 0.8% agarose gel, purified, digested with NcoI and HindIII, and inserted into NcoI/HindIII site of pTrc99A. The resulting plasmid was designated pO251.

Protein Expression in E. coli cells

Cells of E. coli containing plasmid pHE84 were grown at 37 °C to A600 of 0.6 to 0.8, and isopropyl-beta -D-thiogalactopyranoside was added to a final concentration of 1 mM to induce the expression of the ORF-2 product. After induction for 3 h the cells were collected by centrifugation and subjected to electrophoresis according to the standard method of Laemmli (15).

Preparation of Crude Homogenate of the Cells

E. coli cells were grown into late exponential phase in Luria-Bertani medium. The cells were centrifuged, suspended in a solution of 25 mM Tris-HCl buffer, pH 7.7, containing 1 mM EDTA and 10 mM 2-mercaptoethanol (2 ml/g of wet cells), and disrupted with a Branson sonifier.

Methyltransferase Assay and Product Analysis

The incubation mixture contained, in a total volume of 0.5 ml, 0.2 ml of crude homogenate of E. coli cells, 0.1 M Tris-HCl buffer, pH 8.0, 10 mM MgCl2, 10 mM dithiothreitol, 0.4% Triton X-100, 0.5 nmol of FPP, and 0.5 nmol of 1,4-dihydroxy-2-naphthoic acid in 1 µl of ethanol-diethyl ether (2:1, v/v). In addition, 0.47 nmol of [1-14C]IPP with 5 nmol of nonlabeled SAM or 0.5 nmol of nonlabeled IPP with 0.8 nmol of 14C-SAM were added as substrates in reaction mixtures. After incubation at 37 °C for 1 h, the reaction was stopped by addition of 0.5 ml of 0.1 M acetic acid in methanol. The reaction products were extracted with pentane (2 × 2 ml), analyzed by reversed phase TLC with a solvent system of acetone/water (19:1). The positions of authentic standards were visualized with iodine vapor, and the distribution of radioactivity was detected by autoradiography with a Fuji BAS 1000 Mac bioimage analyzer.


RESULTS

Sequence of the ORF-2 Product

Homology search revealed that the predicted amino acid sequence of ORF-2 contains the consensus sequence of some methyltransferases that require SAM as substrate (Fig. 2) (16).


Fig. 2. Comparison of amino acid sequence of ORF-2 and other methyltransferases and related proteins. The amino acids marked "#" represent the methyltransferase consensus sequences designated Regions I-III proposed by Ingrosso et al. (16). ORF2, this work. GerC2, product encoded by one of the genes in B. subtilis gerC cluster involved in spore germination (10); O251, unidentified E. coli protein (12); UbiG, 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone methyltransferase of E. coli (11); COQ3, 3,4-dihydroxy-5-hexaprenylbenzoate methyltransferase of S. cerevisiae (19); DauC, aklanonic acid methyltransferase of Streptomyces sp. strain C5 (17); PmtA, phosphatidylethanolamine methyltransferase of R. sphaeroides (18).
[View Larger Version of this Image (80K GIF file)]

DauC (27% identity) of Streptomyces sp. strain C5 catalyzes the methyl esterification of aklanonic acid in daunomycin biosynthesis (17). E. coli UbiG (22% identity) catalyzes SAM-dependent methylation of 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone, the terminal step in the ubiquinone biosynthesis (11). Rhodobacter sphaeroides PmtA (27% identity) catalyzes the N-methylation of phosphatidylethanolamine (18). COQ3 (17% identity) catalyzes the transfer of a methyl group from SAM to the 3-hydroxyl of 3,4-dihydroxy-5-hexaprenylbenzoate in ubiquinone biosynthesis (19). There are two unidentified genes, gerC2 of B. subtilis (10) and o251 of E. coli (12), whose predicted protein products showed 69 and 37% identity to the ORF-2 product, respectively.


Fig. 3. Overexpression of the ORF-2 gene. E. coli JM109 harboring pHE84 was incubated without (lane 2) or with isopropyl-beta -D-thiogalactopyranoside (lane 3). E. coli JM109 harboring pTrc99A was incubated as the control (lane 1). Total proteins were electrophoresed on SDS-polyacrylamide gel. The protein markers are indicated on the left. The ORF-2 gene product is shown by an arrowhead.
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Construction of Expression Vector of ORF-2

To identify the ORF-2 product, the NcoI-HindIII fragment of the PCR product containing ORF-2 was inserted in pTrc99A, and the resulting plasmid was named as pHE84, which was transformed into E. coli JM109 for expression. E. coli JM109 carrying pHE84 overproduced the ORF-2 product by induction with isopropyl-beta -D-thiogalactopyranoside (Fig. 3).

Methyltransferase Assay

The crude homogenate of E. coli JM109 harboring pHE84, which contained the ORF-2 products overproduced, was incubated with 0.47 nmol of [1-14C]IPP with 5 nmol of nonlabeled SAM or 0.5 nmol of nonlabeled IPP with 0.8 nmol of 14C-SAM. After incubation the products were extracted with pentane and chromatographed on a reversed phase LKC-18 plate with a solvent system of acetone/water (19:1, v/v) (Fig. 4).


Fig. 4. TLC radiochromatograms of the pentane extracts of the products formed by the incubation with the homogenate of E. coli JM109/pHE84. a, the crude homogenate of E. coli JM109 harboring pHE84 was incubated in the mixture as described under "Experimental Procedures" with 0.5 nmol of nonlabeled IPP and 0.8 nmol of 14C-SAM (lane 3) or 0.47 nmol of [1-14C]IPP and 5 nmol of nonlabeled SAM (lane 4). E. coli JM109 harboring pTrc99A was incubated under the same condition with 0.5 nmol of nonlabeled IPP and 0.8 nmol of 14C-SAM (lane 1) or 0.47 nmol of [1-14C]IPP and 5 nmol of nonlabeled SAM (lane 2). The positions of authentic prenyl alcohols and MKs co-chromatographed are indicated on the left and right lanes, respectively. Orig., origin; S.F., solvent front. b, time course of MK-8 formation in the incubation with 0.5 nmol of nonlabeled IPP and 0.8 nmol of 14C-SAM.
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Both the autoradiograms of the products derived from the reaction with [1-14C]IPP (lane 4) and those derived from the reaction with 14C-SAM (lane 3) showed a major radioactivity spot coinciding with MK-8. The radioactivity of the spot of MK-8 increased with the reaction time as shown in Fig. 4b. These facts clearly indicate that the homogenate of E. coli cells harboring pHE84 shows 2-octaprenyl-1,4-naphthoquinone methyltransferase activity, which transfers a methyl group from SAM to synthesize MK-8.

Another major radioactivity spot coinciding with farnesol in lane 4 seems to be the product synthesized by endogenous IPP isomerase, FPP synthase, and a phosphatase of the host cells. When a similar incubation was carried out with the addition of purified FPP synthase of B. stearothermophilus (20), this spot became more distinct (data not shown).

Complementation of the E. coli mutant

The E. coli mutant AN70 cells transformed with pO251 were grown on M9 medium plates. The E. coli AN70 cells harboring pO251 grew well on an M9 medium plate and formed colonies (see Fig. 6). In contrast, AN70 could not grow on this medium.


Fig. 6. Complementation of the E. coli mutant AN70. a, AN70. b, AN70 harboring pO251 were grown on M9 medium plates at 37 °C.
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DISCUSSION

The biosynthetic pathway of MKs has been studied in some detail (21). In B. stearothermophilus the prenyl side chain of MK-7 is derived from HepPP, which is synthesized by HepPP synthase. The gene region of the HepPP synthase has been previously cloned and sequenced, showing three open reading frames, ORF-1, ORF-2, and ORF-3. The structural genes of HepPP synthase have been identified to be ORF-1 and ORF-3, designated heps-1 and heps-2, respectively (9). ORF-2 encodes a protein with a molecular weight of 27,132, which has similar sequences to those of some methyltransferases. In this study, we directly identified ORF-2 as the gene encoding 2-heptaprenyl-1,4-naphthoquinone methyltransferase by examining the enzymatic activity of its product.

There are at least seven enzymes that are involved in the biosynthesis of MK as shown in Fig. 1. Methylation of 2-heptaprenyl-1,4-naphthoquinone is the final step in the biosynthesis of MK-7. The genes encoding the enzymes for the other five steps in menaquinone biosynthesis have been identified so far, and four of them have been shown to be in a cluster (menB, menC, menD, and menE) in E. coli (2). A similar cluster has been also found in B. subtilis (6), although menC has not yet been identified. However, the 2-heptaprenyl-1,4-naphthoquinone methyltransferase gene has not yet been cloned. The ORF-2 of B. stearothermophilus, which is identified as the 2-heptaprenyl-1,4-naphthoquinone methyltransferase gene of this bacterium, is located between the genes encoding the two peculiar components of HepPP synthase. Thus we designate this gene menG.

Fig. 2 shows the comparison of amino acid sequence between 2-heptaprenyl-1,4-naphthoquinone methyltransferase, MenG of B. stearothermophilus, and other methyltransferases. B. stearothermophilus 2-heptaprenyl-1,4-naphthoquinone methyltransferase and the unidentified E. coli protein encoded by o251 share a 37% amino acid sequence identity. Daniels et al. have indicated that ubiE is genetically mapped to the region from 84.5 to 86.5 min on the E. coli chromosome, but they have not identified the gene (12). In this region there are 82 open reading frames including o251, which is located at 86 min on the E. coli chromosome. Of particular interest is the fact that UbiE catalyzes SAM-dependent methylation of 2-octaprenyl-6-methoxy-1,4-benzoquinone, the latter step in the biosynthesis of ubiquinone (21). From the map position and its sequence similarity to that of some methyltransferases, o251 seems to correspond to ubiE (Fig. 5). E. coli produces both of the isoprenoid quinones of ubiquinone-8 and MK-8. An E. coli mutant AN70 (ubiE), which is deficient of a specific methyltransferase in the ubiquinone biosynthetic pathway, has also been shown to be completely devoid of MK and contain a high level of demethylmenaquinone-8 (22). Our complementation experiments with the mutant AN70 harboring pO251, which contains o251 of E. coli in pTrc99A, showed growth restoration of the E. coli mutant on a minimal medium (Fig. 6). These results suggest that E. coli UbiE was encoded by o251 in E. coli. The methylation of demethyl-MK is chemically very similar to that of 2-octaprenyl-6-methoxy-1,4-benzoquinone catalyzed by UbiE, because in both reactions the methyl group is transferred to the 1,4-quinoid ring systems at position C-3. It is therefore interesting to examine whether ubiE encodes the methyltransferase that catalyzes the transfer of methyl groups both to 2-octaprenyl-6-methoxy-1,4-benzoquinone and to 2-octaprenyl-1,4-naphthoquinone.


Fig. 5. Methylation reactions and intermediates in the biosynthetic pathway of MK and ubiquinone. Italics show the genetic loci in mutant strains of each methylation step. The length of the isoprenoid side chain (n) varies depending on the species. SAH, S-adenosylhomocysteine; 1, 2-polyprenyl-6-methoxy-1,4-benzoquinone; 2, 2-polyprenyl-3-methyl-6-methoxy-1,4-benzoquinone; 3, 2-polyprenyl-3-methyl-5-hydroxy-6methoxy-1,4-benzoquinone.
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Because MK-7 is the dominant MK in B. stearothermophilus, the ORF-2 product should be assigned as 2-heptaprenyl-1,4-naphthoquinone methyltransferase. Thus, we propose to designate this gene menG. In our in vitro experiments the cell homogenate of E. coli harboring pHE84 synthesized MK-8, which has the prenyl side chain one isoprene unit longer than that of B. stearothermophilus. This fact indicates that the substrate specificity of the methyltransferase is not very stringent for the prenyl side chain of demethyl-MK. This is similar to the specificities of UbiA of E. coli (23) and of COQ2 of Saccharomyces cerevisiae (24), which are 4-hydroxy-benzoate-octaprenyl transferase and 4-hydroxy-benzoate-hexaprenyl transferase in ubiquinone biosynthesis, respectively. Similarly, the 1,4-dihydroxy-2-naphthoate-octaprenyl transferase of Micrococcus luteus has been shown to have such a wide specificity with respect to prenyl diphosphate substrates (25).


FOOTNOTES

*   This work was supported by Grants-in-aid for Scientific Research 06240102 and 07680620 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. The article must therefore be 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 GenBankTM/EMBL Data Bank with accession number(s) D87054[GenBank].


   To whom correspondence should be addressed. K. Ogura: Tel.: 81-22-217-5621; Fax: 81-22-217-5620; E-mail: ogura{at}icrs.tohoku.ac.jp. T. Koyama: Tel.: 81-22-217-7271; Fax: 81-22-217-7293; E-mail: koyama{at}icrs.tohoku.ac.jp.
1   The abbreviations used are: HepPP, heptaprenyl diphosphate; MK, menaquinone; IPP, isopentenyl diphosphate; FPP, (E,E)-farnesyl diphosphate; PCR, polymerase chain reaction; ORF, open reading frame; SAM, S-adenosyl-L-methionine; 14C-SAM, S-adenosyl-L[methyl-14C]methionine.

ACKNOWLEDGEMENTS

We are grateful to Dr. G. Unden and Dr. I. G. Young for suppling the bacterial strain AN70 and Chika Ishida for cooperation in the construction of pO251.


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