(Received for publication, February 10, 1997)
From the 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.
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).
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.
[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.
Restriction enzyme digestion,
transformation, and other standard molecular biology techniques were
carried out as described by Sambrook et al. (14).
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.
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 To introduce an NcoI site at 5 Cells of E. coli containing plasmid pHE84 were grown at 37 °C to
A600 of 0.6 to 0.8, and
isopropyl- 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.
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.
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).
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.
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- 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).
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).
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.
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.
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).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D87054[GenBank]. 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.
Bio Research Laboratory,
Institute for Chemical Reaction Science,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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)]
Materials
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.
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.
-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).
Sequence of the ORF-2 Product
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)]
Fig. 3.
Overexpression of the ORF-2 gene. E. coli JM109 harboring pHE84 was incubated without (lane
2) or with isopropyl--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.
[View Larger Version of this Image (84K GIF file)]
-D-thiogalactopyranoside (Fig. 3).
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.
[View Larger Version of this Image (41K GIF file)]
Fig. 6.
Complementation of the E. coli
mutant AN70. a, AN70. b, AN70 harboring
pO251 were grown on M9 medium plates at 37 °C.
[View Larger Version of this Image (37K GIF file)]
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.
[View Larger Version of this Image (11K GIF file)]
*
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.
¶
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.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.