J OURNAL OF B ACTERIOLOGY, 0021-9193/98/$04.00ϩ0Mar.1998,p.1578–1581Vol.180,No.6
Copyright©1998,American Society for Microbiology
Molecular Cloning,Expression,and Characterization of the Genes Encoding the Two Essential Protein Components of
Micrococcus luteus B-P26Hexaprenyl
Diphosphate Synthase
NAOTO SHIMIZU,TANETOSHI KOYAMA,*AND KYOZO OGURA
Institute for Chemical Reaction Science,Tohoku University,
Aoba-ku,Sendai980-8577,Japan
Received11August1997/Accepted30December1997
The structural genes encoding the two essential components A and B of hexaprenyl diphosphate synthase,
which produce the precursor of the prenyl side chain of menaquinone-6,were cloned from Micrococcus luteus
B-P26.
Hexaprenyl diphosphate synthase(EC2.5.1.33)(HexPS) catalyzes condensation of three molecules of isopentenyl di-phosphate with farnesyl diphosphate(FPP)to afford(all-E)-hexaprenyl diphosphate(HexPP;C30),the precursor of the prenyl side chain of menaquinone-6.HexPS of Micrococcus luteus B-P26(2)and the heptaprenyl diphosphate(C35)syn-thase(EC2.5.1.30)(HepPS)of Bacillus subtilis(16)are unique because they each consist of two dissociable components;the former are designated A and B(2),and the latter are desig-nated I and II(4).The two components have no prenyltrans-ferase activity unless they are combined(4).These two-com-ponent systems distinguish the medium chain(all-E)-prenyl diphosphate synthases from the other prenyltransferases which have homodimeric structures and catalyze the synthesis of shorter-or longer-chain prenyl diphosphates such as farnesyl, octaprenyl,and undecaprenyl diphosphates(13,14).
The genes for the HepPS of Bacillus stearothermophilus con-sist of two cistrons encoding different components with molec-ular masses of25kDa(component IЈ)and36kDa(component IIЈ),respectively(
6).Zhang et al.(19)have recently identified two of the proteins(GerC1and GerC3)encoded by the gerC locus of B.subtilis(12)as the dissociable heteromeric compo-nents I and II of the HepPS of the bacterium(4).
In order to study the significance and mechanism of the dissociable two-component systems of medium-chain prenyl diphosphate synthases,we cloned the HexPS genes of M.luteus B-P26and compared the deduced amino acid sequences to the corresponding subunits of heptaprenyl diphosphate synthases of the Bacillus species.
To amplify DNA fragments that might have typical motifs for prenyltransferases,we synthesized seven degenerate oligo-nucleotide primers designed on the basis of conserved amino acid regions of prenyltransferases(8,9).An amplified product of approximately500bp(designated B500)was obtained by PCR with the pair P1and N3as primers[P1,5Ј-GG(A,T,C)G G(A,T,C)AA(A,G)CGTA(A,T)TCGTCCTTTA-3Ј;N3,5Ј-A TCTAAAATATCATC(C,T)TG(A,T)AT(C,T)TG(A,G)AA-3Ј]with the genomic DNA template of M.luteus B-P26.The amino acid sequence deduced from the nucleotide sequence of B500contained the typical prenyltransferase motif DDXXD (1,8)and showed61%identity with the corresponding region of    B.stearothermophilus FPP synthase(FPS)(8).This 500-bp PCR fragment was used for screening
the prenyltrans-ferase gene(s)from an M.luteus B-P26genomic library pre-pared in Escherichia coli JM109harboring plasmids of pUC119 with inserts of4-to8-kb DNA fragments.Among6,000indi-vidual colonies,a single positive clone was found.This clone was further purified,and the plasmid,designated pFP00, was shown to carry a10-kb DNA insert from M.luteus B-P26. Product analysis of a prenyltransferase with increased activity in the cell-free homogenate of the clone indicated that pFP00 contained the FPS gene(fps)of M.luteus B-P26(Fig.1A). Southern blot analysis with the radiolabeled B500fragment, which was revealed to be a partial fragment of fps,gave a strong band at7.5kb and a faint band at4.2kb in the lane with Eco RI-digested genomic DNA(data not shown).We assigned the former,strong band to the FPS gene and the latter to the other prenyltransferase gene(2,10)having some similarity.A subgenomic library of M.luteus B-P26was prepared from4to 6kb of Eco RI-digested DNA,which contains the fragment weakly hybridizable with B500and excludes the fps gene.Col-ony hybridization with B500yielded3positive colonies carry-ing the same5.7-kb DNA fragment among1,200colonies.The PCR primers were also used as probes tofind coinciding pos-itive signals.Cell-free homogenates of the clones showed evi-dently higher prenyl diphosphate synthase activities than en-dogenous prenyl diphosphate synthase activities of the li cells,and analysis of the reaction products by thin-layer chromatography(TLC)indicated that the clone produced HexPP along with some shorter-chain interm
ediate prenyl di-phosphates(Fig.1B).Then the clone,designated pHX00,was subjected to a deletion experiment and sequence determina-tion to obtain a clone carrying pHX06with a2.4-kb DNA insert(Fig.2)expressing HexPS activity.
Analysis of the pHX06nucleotide sequence showed three consecutive open reading frames,tentatively designated ORF1, ORF2,and ORF3.In order to determine the structural genes corresponding to HexPS,which had been shown to consist of two dissociable components(2),we prepared three plasmids, pREG1,pREG2,and pREG3S,having one of the three clones (Fig.2),and examined the enzymatic activity of their protein products expressed li cells.Although none of the cell-
*Corresponding author.Mailing address:Institute for Chemical Reaction Science,Tohoku University,Katahira2-1-1,Aoba-ku,Sendai 980-8577,Japan.Phone:81-22-217-5621.Fax:81-22-217-5620.E-mail: hoku.ac.jp.
clone
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free homogenates of the three clones showed any prenyltrans-ferase activity alone (Fig.1C),a significant level of prenyltrans-ferase activity was observed when the homogenates of the pREG1an
d pREG3S transformants were mixed together.As shown in Fig.1C,the mixture of the homogenates of the pREG1and pREG3S transformants gave a major spot of C 30-polyprenol along with some amounts of shorter-chain prenols derived from the corresponding intermediate prenyl diphos-phates.These results indicate that ORF1and ORF3encode the two essential components of the HexPP synthase.We named the genes hexs-a and hexs-b ,respectively.Comparison of the deduced amino acid sequences of the two components encoded by hexs-a and hexs-b with those for the other medium-chain prenyl diphosphate synthases indicated that the Hexs-b protein (component B)shows 38,41,and 31%identity to component II Ј(Heps-2)of HepPS of B.stearother-mophilus (10),component II (GerC)of HepPS of B.subtilis (19),and HexPS visiae (1),respectively.On the other hand,Hexs-a (component A)has only 14of 143amino acid residues identical to components I and I Јof the HepPSs from the two Bacillus species (10%identity,as indicated by asterisks in Fig.3),while there is 31%identity (69of 220residues,
as
FIG.1.Autoradiograms of TLC of the prenyl alcohols obtained by enzymatic hydrolysis (3)of the products formed by the incubations of [1-14C]isopentenyl diphosphate and dimethylallyl diphosphate or FPP with the cell-free homogenates li transformants.(A)Incubations with dimethylallyl dipho
sphate.Products were derived li JM 109/pUC119(control)(lane 1)li JM 109/pFP00(lane 2).(B)Incubations with FPP.Products were derived li JM 109/pUC119(control)(lane 1)li JM 109/pHX00(lane 2).(C)Incubations with FPP.Products were derived li JM 109/pUC119(control)(lane 1),E.coli JM 109/pREG1(lane 2),E.coli JM 109/pREG2(lane 3),E.coli JM 109/pREG3S (lane 4),the mixture li JM 109/li JM 109/pREG2(lane 5),the mixture li JM 109/li JM 109/pREG3S (lane 6),the mixture li JM 109/li JM 109/pREG3S (lane 7),li JM 109/pHX06(lane 8).Each extract was analyzed by reversed phase TLC (type LKC-18;Whatman)with solvent systems of acetone-water (9:1[B]or 19:1[A and C]).Arrowheads indicate the positions of authentic prenyl alcohols as follows:C 10,geraniol;C 15,(all-E )-farnesol;C 20,(all-E )-geranylgeraniol;C 30,(all-E )-hexaprenol;C 45,(all-E )-nonaprenol (solanesol);C 55,beturaprenol-55,C 60,beturaprenol-60;Ori.,origin;S.F.,solvent
front.
FIG.2.Schematic diagram of plasmids prepared.Only the inserted DNA regions in each plasmid are ill
ustrated.Thick lines in each plasmid indicate the chromosomal DNA from M.luteus B-P 26,and thin lines indicate parts of the vector,pUC119.Open arrows show the three open reading frames found in the DNA region responsible for expression of HexPS.Abbreviations:H,Hin dIII;P,Pst I;Hi,Hin cII;X,Xba I;B,Bam HI;K,Kpn I;S,Sac I;E,Eco RI;E14,Eco T14I;N,Nru I.
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indicated by shaded boxes in Fig.3)between the latter two components.Moreover,component A of HexPS (Hexs-a)is shorter by about 100-amino-acid residues at the C-terminal side than the corresponding component I or I Јof HepPS.The three conserved regions in the structure of component A,as indicated by asterisks in Fig.3,may participate in the dynamic interaction,as observed biochemically,between the two disso-ciable components forming a catalytically active complex in the presence of substrates and Mg 2ϩ(17,18).
The protein encoded by hexs-b contains seven conserved regions,including the two aspartate-rich m
otifs DDXXD,which have been proved in several site-directed mutagenesis studies of FPSs to be essential for their catalytic function (5,7,11,15).Thus,this component is reasonably considered to carry substantial sites for substrate binding and catalysis,while the other component seems to play an auxiliary but essential role for expression of catalytic function as medium-chain prenyl diphosphate synthase.
The primary structure of the protein encoded by ORF2in this work shows a relatively high identity (60%)to that of the protein encoded by the gene that is located between heps -1and the heps -2of B.stearothermophilus (6).Koike-Takeshita et al.(7)have recently identified the gene product of B.stearother-mophilus as MenG,2-heptaprenyl-1,4-naphthoquinone meth-yltransferase,and proposed the presence of a novel gene clus-ter participating in menaquinone-7biosynthesis.Thus,the gene located between hexs-a and hexs-b is likely to encode the equivalent enzyme,2-hexaprenyl-1,4-naphthoquinone methyl-transferase,of M.luteus B-P 26,thereby forming a similar gene cluster participating in menaquinone-6biosynthesis in this bac-terium.
Ashby and Edwards (1)have isolated the HexPS gene of Saccharomyces cerevisiae ,which is able to complement a yeast mutant in coenzyme Q biosynthesis.This gene encodes a 473-amino-acid protein having highly conserved domains charac-teristic of prenyl diphosphate synthases.However,it
is not known whether the yeast protein acts as HexPS by itself or in association with another gene product corresponding to com-ponent A of HexPS of M.luteus B-P 26.We have not found any proteins that have conserved sequences in Hexs-a among the open reading frames of visiae whole-genome data-base.It would be interesting to investigate whether the eukary-otic HexPS has a two-component system similar to that of the prokaryotic HexPS described in this study.
Nucleotide sequence accession numbers.The nucleotide se-quences reported appear in the DDBJ,EMBL,and GenBank nucleotide sequence databases under accession no.AB003188
for the fps gene and AB003187for the DNA containing hexs-a and hexs-b.
This work was supported by grants-in-aid for scientific research no.06240102and 07680620from the Ministry of Education,Science,and Culture,Japan.
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