Dimer Formation of Octaprenyl-diphosphate Synthase (IspB) Is Essential for Chain Length Determination of Ubiquinone
2001; Elsevier BV; Volume: 276; Issue: 11 Linguagem: Inglês
10.1074/jbc.m007472200
ISSN1083-351X
AutoresTomohiro Kainou, Kazunori Okada, Kengo Suzuki, Tsuyoshi Nakagawa, Hideyuki Matsuda, Makoto Kawamukai,
Tópico(s)Biochemical Acid Research Studies
ResumoUbiquinone (Q), composed of a quinone core and an isoprenoid side chain, is a key component of the respiratory chain and is an important antioxidant. In Escherichia coli, the side chain of Q-8 is synthesized by octaprenyl-diphosphate synthase, which is encoded by an essential gene, ispB. To determine how IspB regulates the length of the isoprenoid, we constructed 15 ispB mutants and expressed them inE. coli and Saccharomyces cerevisiae. The Y38A and R321V mutants produced Q-6 and Q-7, and the Y38A/R321V double mutant produced Q-5 and Q-6, indicating that these residues are involved in the determination of chain length. E. colicells (ispB::cat) harboring an Arg-321 mutant were temperature-sensitive for growth, which indicates that Arg-321 is important for thermostability of IspB. Intriguingly, E. coli cells harboring wild-type ispB and the A79Y mutant produced mainly Q-6, although the activity of the enzyme with the A79Y mutation was completely abolished. When a heterodimer of His-tagged wild-type IspB and glutathioneS-transferase-tagged IspB(A79Y) was formed, the enzyme produced a shorter length isoprenoid. These results indicate that although the A79Y mutant is functionally inactive, it can regulate activity upon forming a heterodimer with wild-type IspB, and this dimer formation is important for the determination of the isoprenoid chain length. Ubiquinone (Q), composed of a quinone core and an isoprenoid side chain, is a key component of the respiratory chain and is an important antioxidant. In Escherichia coli, the side chain of Q-8 is synthesized by octaprenyl-diphosphate synthase, which is encoded by an essential gene, ispB. To determine how IspB regulates the length of the isoprenoid, we constructed 15 ispB mutants and expressed them inE. coli and Saccharomyces cerevisiae. The Y38A and R321V mutants produced Q-6 and Q-7, and the Y38A/R321V double mutant produced Q-5 and Q-6, indicating that these residues are involved in the determination of chain length. E. colicells (ispB::cat) harboring an Arg-321 mutant were temperature-sensitive for growth, which indicates that Arg-321 is important for thermostability of IspB. Intriguingly, E. coli cells harboring wild-type ispB and the A79Y mutant produced mainly Q-6, although the activity of the enzyme with the A79Y mutation was completely abolished. When a heterodimer of His-tagged wild-type IspB and glutathioneS-transferase-tagged IspB(A79Y) was formed, the enzyme produced a shorter length isoprenoid. These results indicate that although the A79Y mutant is functionally inactive, it can regulate activity upon forming a heterodimer with wild-type IspB, and this dimer formation is important for the determination of the isoprenoid chain length. ubiquinone geranylgeranyl diphosphate farnesyl diphosphate isopentenyl diphosphate polymerase chain reaction glutathioneS-transferase nitrilotriacetic acid high pressure liquid chromatography Ubiquinone (Q)1 is an essential component of the electron transport system associated with aerobic growth and oxidative phosphorylation in many organisms. It also has been reported that ubiquinone has an important role as an antioxidant in Escherichia coli (1Søballe B. Poole R.K. Microbiology ( Read. ). 2000; 146: 787-796Crossref PubMed Scopus (84) Google Scholar),Schizosaccharomyces pombe (2Suzuki K. Okada K. Kamiya Y. Zhu X. Tanaka K. Nakagawa T. Kawamukai M. Matsuda H. J. Biochem. ( Tokyo ). 1997; 121: 496-505Crossref PubMed Scopus (80) Google Scholar, 38Uchida N. Suzuki K. Saiki R. Kainou T. Tanaka T. Matsuda H. Kawamukai M. J. Bacteriol. 2000; 182: 6933-6939Crossref PubMed Scopus (63) Google Scholar), Saccharomyces cerevisiae (3Do T.Q. Schultz J.R. Clarke C.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7534-7539Crossref PubMed Scopus (143) Google Scholar), and mammalian cells (4Frei B. Kim M.C. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4879-4883Crossref PubMed Scopus (561) Google Scholar). Furthermore, it was elegantly shown that ubiquinone (or menaquinone) accepts electrons generated by the formation of protein disulfide in E. coli(5Bader M. Muse W. Ballou D.P. Gassner C. Bardwell J.C.A. Cell. 1999; 98: 217-227Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). Thus, multiple functions of ubiquinone have been proposed. The ubiquinone biosynthetic pathway, which is comprised of 10 steps including methylation, decarboxylation, hydroxylation, and isoprenoid transfer, has been studied genetically in respiratory-deficient mutants of E. coli and S. cerevisiae (6Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google Scholar). Each of these organisms has a specific isoprenoid side chain length as part of the ubiquinone molecule, e.g. Q-6 for S. cerevisiae, Q-8 for E. coli, Q-9 for rat, and Q-10 for S. pombe and human. For this reason, ubiquinone species have been used for classification in microbial taxonomy (7Collins M.D. Jones D. Microbiol. Rev. 1981; 45: 316-354Crossref PubMed Google Scholar). The length of the side chain of ubiquinone is precisely defined by the action of polyprenyl-diphosphate synthases, but not by 4-hydroxybenzoate-polyprenyl-diphosphate transferases, which catalyze the condensation of 4-hydroxybenzoate and polyprenyl diphosphate (8Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google Scholar). When various polyprenyl-diphosphate synthase genes, such as the mutant GGPP synthase gene from Sulfolobus acidocaldarius, the hexaprenyl-diphosphate synthase gene (COQ1) from S. cerevisiae, the heptaprenyl-diphosphate synthase gene fromHaemophilus influenzae, the octaprenyl-diphosphate synthase gene (ispB) from E. coli, the solanesyl-diphosphate synthase gene (sdsA) fromRhodobacter capsulatus, and the decaprenyl-diphosphate synthase gene (ddsA) from Gluconobacter suboxydans, are expressed in an S. cerevisiae COQ1mutant, each transformant produced mainly Q-5, -6, -7, -8, -9, and -10, respectively (9Okada K. Kainou T. Matsuda H. Kawamukai M. FEBS Lett. 1998; 431: 241-244Crossref PubMed Scopus (72) Google Scholar). When COQ2, which encodes 4-hydroxybenzoate-hexaprenyl-diphosphate transferase in S. cerevisiae, was expressed in an E. coli ubiA mutant cell line, the transformant produce Q-8, but not Q-6 (10Suzuki K. Ueda M. Yuasa M. Nakagawa T. Kawamukai M. Matsuda H. Biosci. Biotechnol. Biochem. 1994; 58: 1814-1819Crossref PubMed Scopus (65) Google Scholar). These results indicate that prenyl-diphosphate synthase determines the chain length of ubiquinone and that 4-hydroxybenzoate-polyprenyltransferases can accept the various isoprenoid chains as a substrate. In E. coli, ispB is an essential gene, responsible for the biosynthesis of both ubiquinone and menaquinone (11Okada K. Minehira M. Zhu X. Suzuki K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 3058-3060Crossref PubMed Google Scholar, 12Søballe B. Poole R.K. Microbiology ( Read. ). 1999; 145: 1817-1830Crossref PubMed Scopus (181) Google Scholar). An E. coli ubiA mutant, which does not produce Q-8, is not able to grow on a non-fermentable carbon source, but can grow on glucose (10Suzuki K. Ueda M. Yuasa M. Nakagawa T. Kawamukai M. Matsuda H. Biosci. Biotechnol. Biochem. 1994; 58: 1814-1819Crossref PubMed Scopus (65) Google Scholar). However, an E. coli ubiA − menA − mutant, which lacks both ubiquinone and menaquinone biosynthesis genes, can grow only when a small amount of Q-8 is still produced by leakiness of the mutations. Thus,ubiA − menA − mutants with an absolute lack of production of Q-8 and menaquinone-8 cannot be isolated. Long-chain polyprenyl-diphosphate synthases (C40, C45, and C50) catalyze the condensation of FPP, which acts as a primer, and IPP to produce each prenyl diphosphate with various chain lengths. These enzymes possess seven conserved regions including two DDXXD motifs that are binding sites for the substrates in association with Mg2+ (13Koyama T. Obata S. Osabe M. Takeshita A. Yokoyama K. Uchida M. Nishino T. Ogura K. J. Biochem. ( Tokyo ). 1993; 113: 355-363Crossref PubMed Scopus (123) Google Scholar, 14Chen A. Kroon P.A. Poulter C.D. Protein Sci. 1994; 3: 600-607Crossref PubMed Scopus (222) Google Scholar). Short-chain polyprenyl-diphosphate synthases (C15 and C20), such as FPP and GGPP synthases, have been identified in organisms ranging from bacteria to mammals (15Kainou T. Kawamura K. Tanaka K. Matsuda H. Kawamukai M. Biochim. Biophys. Acta. 1999; 1437: 333-340Crossref PubMed Scopus (40) Google Scholar), and the mechanisms that determine the chain length have been reported (16Ohnuma S.-i. Hirooka K. Tsuruoka N. Yano M. Ohto C. Nakane H. Nishino T. J. Biol. Chem. 1998; 273: 26705-26713Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Site-directed mutagenesis of farnesyl-diphosphate synthases from Bacillus stearothermophilus (16Ohnuma S.-i. Hirooka K. Tsuruoka N. Yano M. Ohto C. Nakane H. Nishino T. J. Biol. Chem. 1998; 273: 26705-26713Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) and certain avian species (17Tarshis L.C. Proteau P.J. Kellogg B.A. Sacchettini J.C. Poulter C.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15018-15023Crossref PubMed Scopus (315) Google Scholar) and geranylgeranyl-diphosphate synthase from S. acidocaldarius(16Ohnuma S.-i. Hirooka K. Tsuruoka N. Yano M. Ohto C. Nakane H. Nishino T. J. Biol. Chem. 1998; 273: 26705-26713Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) was used to determine which amino acids are important for the determination of chain length of short-chain prenyl diphosphates. These amino acids were those at the fourth and fifth positions before an aspartate-rich motif in region II or one amino acid at the fifth position before this motif and two amino acids in region II. Recently, we reported that substitution of glycine for alanine before the first DDXXD motif in decaprenyl-diphosphate synthase allowed the enzyme to synthesize products with longer chain lengths (18Okada K. Kainou T. Tanaka K. Nakagawa T. Matsuda H. Kawamukai M. Eur. J. Biochem. 1998; 255: 52-59Crossref PubMed Scopus (83) Google Scholar). Thus, the fifth amino acid before region II of long-chain polyprenyl-diphosphate synthases plays an important role in the mechanism of chain length determination (18Okada K. Kainou T. Tanaka K. Nakagawa T. Matsuda H. Kawamukai M. Eur. J. Biochem. 1998; 255: 52-59Crossref PubMed Scopus (83) Google Scholar). Generally, polyprenyl-diphosphate synthases are known to function as a dimer. The medium-chain polyprenyl-diphosphate synthases (C30 and C35) from Micrococcus luteus BP26, B. stearothermophilus, and Bacillus subtilis are composed of heterodimers (19Zhang Y.-W. Koyama T. Ogura K. J. Bacteriol. 1997; 179: 1417-1419Crossref PubMed Google Scholar, 20Shimizu N. Koyama T. Ogura K. J. Bacteriol. 1998; 180: 1578-1581Crossref PubMed Google Scholar). GGPP synthase purified from bovine brain forms a homo-oligomer (150–195 kDa) (21Sagami H. Morita Y. Ogura K. J. Biol. Chem. 1994; 269: 20561-20566Abstract Full Text PDF PubMed Google Scholar). However, the subunit structure of long-chain polyprenyl-diphosphate synthases remains to be determined. Recently, geranyl-diphosphate synthase isolated from spearmint (22Burke C.C. Wildung M.R. Croteau R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13062-13067Crossref PubMed Scopus (183) Google Scholar) was found to form a heterodimer. One subunit has similarity with known prenyltransferases, and the other has similarity with the Arabidopsis GGR protein (23Scolnik P.A. Bartley G.E. Plant Physiol. 1995; 108: 1343Google Scholar), but the aspartate-rich motifs are not conserved. In this study, we describe the mutational analysis of octaprenyl-diphosphate synthase (IspB) from E. coli. From the analysis, we found that IspB forms a homodimer that is important for the determination of isoprenoid chain length. Restriction enzymes and other DNA-modifying enzymes were purchased from Takara Shuzo Co., Ltd., and New England Biolabs, Inc. IPP, (E)-farnesyl diphosphate (all-(E)-FPP), geranylgeraniol, and solanesol (all-(E)-nonaprenol) were purchased from Sigma. [1-14C]IPP (1.96 TBq/mol) was purchased from Amersham Pharmacia Biotech. Kieselgel 60 F254 TLC plates were purchased from Merck. Reversed-phase LKC-18 TLC plates were purchased from Whatman. E. coli strains DH10B and JM109 were used in the general construction of plasmids (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). KO229 (ispB::cat) (11Okada K. Minehira M. Zhu X. Suzuki K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 3058-3060Crossref PubMed Google Scholar), which is theispB-defective mutant of E. coli harboring pKA3 (ispB), was used as a host strain to express IspB mutants and for ubiquinone extraction. YKK6 (COQ1::URA3) (8Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google Scholar), which is the COQ1-defective mutant of S. cerevisiae, was used for complementation analysis and ubiquinone extraction. The plasmids pBluescript KS(−)/SK(+) and YEp13M4 were used as vectors (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar, 25Rose M.D. Broach J.R. Methods Enzymol. 1991; 194: 195-230Crossref PubMed Scopus (214) Google Scholar). The strains and plasmids used in this study are listed in Table I.Table IStrains and plasmids used in this studyStrain or plasmidRelevant characteristicsSource or Ref.StrainE. coliDH10BlacZM1524Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google ScholarE. coliJM109lacI q lacZ M1524E. coli KO229Cmr Spr ispB∷cat; harbors pKA311Okada K. Minehira M. Zhu X. Suzuki K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 3058-3060Crossref PubMed Google ScholarE. coliKO229/35–2Cmr Kmr Apr; harbors pSTVKQKO56 and pG79YThis studyS. cerevisiaeYKK6URA3 + COQ1∷URA38Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google ScholarPlasmidpBluescript KS(−)Apr lacZStratagenepBluescript SK(+)Apr lacZStratageneYEp13M4Apr LEU + 2μm25Rose M.D. Broach J.R. Methods Enzymol. 1991; 194: 195-230Crossref PubMed Scopus (214) Google ScholarpKA3Spr; 3-kbEcoRI fragment including ispB in pCL192011Okada K. Minehira M. Zhu X. Suzuki K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 3058-3060Crossref PubMed Google ScholarpQE31Apr; T5 promoter, His tag, high expression vectorQIAGENpGEX-1Apr; tac promoter, GST tag, high expression vectorAmersham PharmaciapSTVK28Kmr; derived from pSTV28Takara ShuzopKO56Apr; 2.5-kb SspI-HindIII fragment including ispB in pBluescript8Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google ScholarpSA1Apr; 0.3-kb BamHI-EcoRI fragment of 5′-end COQ1 in pBluescript KS(+)8Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google ScholarpQKO56Apr; 1.0-kb BamHI-HindIIIispB gene in pQE31This studypGKO56Apr; 1.0-kb BamHI-XhoIispB gene in pGEX-1XThis studypG79YApr; 1.0-kb BamHI-XhoI A79Y mutant ispB gene in pGEX-1XThis studypSTVKQKO56Kmr; 1.0-kbEcoRI-HindIII fragment from pQKO56 in pSTVK28This studyCm, chloramphenicol; Sp, spectinomycin; Km, kanamycin; Ap, ampicillin; kb, kilobase pair. Open table in a new tab Cm, chloramphenicol; Sp, spectinomycin; Km, kanamycin; Ap, ampicillin; kb, kilobase pair. Site-directed mutagenesis by PCR was performed following the method of Ito et al. (26Ito W. Ishiguro H. Kurosawa Y. Gene ( Amst. ). 1991; 102: 67-70Crossref PubMed Scopus (261) Google Scholar). Four oligonucleotide primers (MUT, R1 (for each mutational primer), T7, and T3) (see Table II) were used in amplifications. pKO56, which contains the open reading frame and downstream region of ispB, was used as template in PCRs. First, PCR was performed with the MUT and T3 primers in one reaction and with the R1 and T7 primers in another. An aliquot of each of the reaction mixtures was mixed in a new tube to form the heteroduplex ispB template, and full-length mutantispB was amplified with the T7 and T3 primers by PCR. This DNA fragment was digested with EcoRI and HindIII and cloned into pBluescript KS(−). This construct was transformed intoE. coli DH10B and KO229 (ispB::cat) for analysis of enzyme activity and ubiquinone production.Table IIPrimers used to construct ispB mutants in this studyMutant namePrimer sequenceL31V5′-CTGATTGATCACTTGGACGT-3′I32V5′-ACTGATTGACCAGCTGGACGTCGGA-3′Y37A5′-TGACGATGTAAGCGCCTAACTGA-3′Y38A5′-GCTGACGATCGCATAGCCTAA-3′Y37A/Y38A5′-CCGCTGACGATCGCAGCGCCTAACTGATT-3′Y38A/R321VCombination of Y38A and R321VY61V5′-CATTTCCCTCGACGCCAACAGCT-3′F75A5′-CGTGTGGATAGCCTCGATCA-3′A79Y5′-GTAGCAGAGTATACGTGTGGAT-3′K170A5′-ACGCGCGGTTGCGCTATAGATA-3′K170G5′-ACGCGCGGTTCCGCTATAGATA-3′K235L5′-CAGCGTCGGTAAACCTTCGTT-3′R321A5′-TTAACGATCGGCTTGAACAGCG-3′R321D5′-TTAACGATCGTCTTGAACAGCG-3′R321V5′-TTAACGATCGACTTGAACAGCG-3′MUT5′-AGTGGAACCTCCGGGCAGCAGCAATTC-3′T35′-AATACGACTCACTATAG-3′T75′-ATTAACCCTCACTAAAG-3′S25′-TCGAATTCTATGAATTTAGAAAAAATC-3′A35′-CGAAGCTTGGCCATGGGCGCGAT-3′QEGST5′-TCGGATCCGATGAATTTAGAAAAAATC-3′ Open table in a new tab E. coliKO229 (ispB::cat) harboring pKA3 (11Okada K. Minehira M. Zhu X. Suzuki K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 3058-3060Crossref PubMed Google Scholar) was transformed with the plasmid containing mutant ispB, which produced transformants that were resistant to spectinomycin and ampicillin. The transformants were subcultured five times in LB medium containing 50 μg/ml ampicillin and plated on LB agar medium containing ampicillin. The resulting colonies were then replicated on LB medium containing ampicillin or spectinomycin. Spectinomycin-sensitive and ampicillin-resistant strains, which had the mutant ispB plasmid, but not pKA3, were selected and used for ubiquinone analysis. To express the mutant ispB genes in S. cerevisiaeYKK6 (COQ1::URA3), aCOQ1-ispB fusion gene was constructed. The S2 and A3 primers were used to amplify the ispB gene by PCR. The fragment was digested with EcoRI and HindIII and cloned into pSA1 (8Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google Scholar), which has 53 amino acids of the Coq1 mitochondrial import signal with the COQ1 promoter. TheBamHI-HindIII fragment also was cloned into the yeast shuttle vector YEp13M4 (25Rose M.D. Broach J.R. Methods Enzymol. 1991; 194: 195-230Crossref PubMed Scopus (214) Google Scholar). YKK6 was transformed with both plasmids by the lithium acetate method (27Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar) and was selected on Synthetic Complete (0.67% (w/v) yeast nitrogen base, 2% (w/v) glucose or 3% (w/v) glycerol, and the appropriate amino acids)−Leu−Ura medium. To overexpress and purify IspB, vectors containing the 6-His or glutathione S-transferase (GST) tag fused to IspB were constructed. The amplifiedBamHI-HindIII fragment containing ispBfrom pKO56 was cloned into pQE31 (QIAGEN Inc.) to yield pQKO56. The amplified BamHI-XhoI fragment containing wild-type or A79Y mutant ispB was cloned into pGEX-1X in which an XhoI linker had been inserted to yield pGKO56 or pG79Y, respectively. The plasmids were transformed into E. coli JM109. Transformants were grown to stationary phase in LB medium containing 50 μg/ml ampicillin, and 10 ml of culture was inoculated into 100 ml of the same medium. The culture was grown at 37 °C for 3 h, and recombinant protein expression was induced with 0.1 mmisopropyl-1-thio-β-d-galactopyranoside. The cells were collected by centrifugation at 2500 × g for 10 min. To purify His-tagged IspB, cells were suspended in 50 mmsodium phosphate, 300 mm NaCl, and 10 mmimidazole and sonicated 10 times for 10 s at 10-s intervals with an ultrasonic disintegrator in an ice bath. Ruptured cells were centrifuged at 15,000 × g for 20 min. The resulting supernatants were added to a Ni2+-nitrilotriacetic acid (NTA) slurry and mixed gently at 4 °C for 60 min. This mixture was loaded onto a column and washed with 50 mm sodium phosphate, 300 mm NaCl, and 20 mm imidazole. The His-IspB protein was eluted with 50 mm sodium phosphate, 300 mm NaCl, and 250 mm imidazole. To purify the GST-IspB protein, cells were suspended in 50 mm Tris-HCl (pH 8.0), 50 mm NaCl, 1 mm EDTA, and 1 mm dithiothreitol (sonication buffer). Cells were ruptured by sonication, and the lysate was mixed with glutathione-Sepharose 4B (Amersham Pharmacia Biotech) at 4 °C for 60 min. This mixture was washed twice with 140 mm NaCl, 2.7 mm KCl, 10 mm sodium phosphate, and 1.8 mm potassium phosphate and then with sonication buffer. The GST-IspB protein was eluted with sonication buffer containing 10 mm reduced glutathione. TheEcoRI-HindIII fragment from pQKO56 was recloned into pSTVK28, which had been converted from expressing chloramphenicol resistance to kanamycin resistance, to yield pSTVKQKO56. KO229 cells harboring pBRA, which expresses IspB containing the mutation R321A, were transformed with pSTVKQKO56 and produced transformants that were resistant to ampicillin and kanamycin. Ampicillin-sensitive and kanamycin-resistant strains were selected following the method described above and transformed with pG79Y, and the strains harboring both plasmids were selected and named KO229/35-2. Ubiquinone extraction was performed by the method described previously (8Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google Scholar, 28Wallace B.J. Young I.G. Biochim. Biophys. Acta. 1977; 461: 84-100Crossref PubMed Scopus (209) Google Scholar). The crude extract of ubiquinone was analyzed by normal-phase TLC with authentic standard Q-10. Normal-phase TLC was carried out on a Kieselgel 60 F254 plate with benzene/acetone (97:3, v/v). The band containing ubiquinone was collected from the TLC plate following UV visualization and extracted with chloroform/methanol (1:1, v/v). Samples were dried and redissolved in ethanol. The purified ubiquinone was further analyzed by HPLC with ethanol as the solvent. Prenyl-diphosphate synthase activity was measured by the method described previously (8Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google Scholar), in which incorporation of [1-14C]IPP into reaction products is detected. E. coli DH10B or KO229 cells, harboring plasmids containing mutantispB, were incubated to a late log phase in LB medium containing appropriate antibiotics at 37 °C. Cells were harvested by centrifugation; suspended in buffer A (100 mm potassium phosphate (pH 7.4), 5 mm EDTA, and 1 mm2-mercaptoethanol); and ruptured by six sonication treatments, each lasting 30 s with 30-s intervals, in an ice bath. After centrifugation of the homogenate, the supernatant was used as a crude enzyme extract. The assay reaction mixture contained 1.0 mmMgCl2, 0.1% (w/v) Triton X-100, 50 mmpotassium phosphate buffer (pH 7.5), 10 μm[1-14C]IPP (specific activity of 0.92 TBq/mol), 5 μm FPP, and 200 μg of crude extract containing the enzyme in a final volume of 0.4 ml. Sample mixtures were incubated for 60 min at 30 °C. Reaction products such as prenyl diphosphates were extracted with 1-butanol-saturated water and hydrolyzed with acid phosphatase (29Fujii H. Koyama T. Ogura K. Biochim. Biophys. Acta. 1982; 712: 716-718Crossref PubMed Scopus (157) Google Scholar). The products of hydrolysis were extracted with hexane and analyzed by reversed-phase TLC with acetone/water (19:1, v/v). Radioactivity on the plate was detected with a BAS1500-Mac imaging analyzer (Fuji Film Co.). The plate was exposed to iodine vapor to detect the spots of the marker prenols. It is known that the side chain length of ubiquinone is determined by the corresponding polyprenyl-diphosphate synthase (8Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google Scholar), but it is not clear how polyprenyl-diphosphate synthase determines this length. To understand the nature of polyprenyl-diphosphate synthase, we analyzed the activity of octaprenyl-diphosphate synthase (IspB), which produces the side chain of Q-8 in E. coli. For this purpose, we constructed 15 IspB mutants by site-directed mutagenesis as shown in Fig. 1 and TableII (primers that were used). Site-directed mutagenesis was performed following the method of Itoet al. (26Ito W. Ishiguro H. Kurosawa Y. Gene ( Amst. ). 1991; 102: 67-70Crossref PubMed Scopus (261) Google Scholar), and the substitutions in all mutantispB genes were confirmed by sequence analysis. E. coli DH10B was transformed with the plasmids containing mutant ispB genes, and the transformants were used in ubiquinone analysis. Because DH10B has the wild-type ispB gene in the form of genomic DNA, the main product is expected to be Q-8. Although most DH10B cells harboring the mutant ispB gene produced Q-8, a number of mutants produced Q-8 with small amounts of Q-6 and Q-7 (data not shown); and interestingly, DH10B harboring the A79Y mutant produced mainly Q-6 (see Fig. 6 A). To detect the actual ubiquinone species produced by the product of the mutant ispB gene, E. coliKO229 (ispB::cat)/pKA3 (ispB), which is defective for the genomic ispB gene, but retainsispB in a plasmid, was transformed with the plasmids containing the mutant ispB genes. KO229, which harbors the mutant ispB genes and has lost the wild-type ispBplasmid (pKA3), was selected as described under “Experimental Procedures.” L31V, I32V, Y38A, Y37A/Y38A, Y38A/R321V, Y61V, F75A, K235L, R321A, R321D, and R321V mutant KO229 strains were obtained; however, Y37A, A79Y, K170G, and K170A mutant KO229 strains could not be isolated. Since ispB is essential for growth of E. coli (11Okada K. Minehira M. Zhu X. Suzuki K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 3058-3060Crossref PubMed Google Scholar), the inability to replace wild-type ispB with these mutants suggested that the Y37A, A79Y, K170G, and K170A mutants do not retain functional activity. The mutants that could complement the loss of the wild-type gene were further analyzed by ubiquinone extraction and analysis (Fig. 2). In the Y38A mutant, Q-7 was mainly produced, with lesser amounts of Q-6 and Q-8 (Fig. 2 D). In the Y37A/Y38A mutant, Q-7 and Q-6 were mainly produced, with a little Q-8 (Fig. 2 E). In the Y38A/R321V and R321V mutants, Q-6 was mainly produced, with a small amount of Q-5 and Q-7 (Fig. 2, F and L, respectively); however, hardly any Q-8 was produced. In the K235L and R321A mutants, Q-8 was mainly produced; however, a minor product (Q-7) was produced at a level that was greater than that with wild-type IspB (Fig. 2, I and J, respectively). These results indicate that Tyr-38, Lys-235, and Arg-321 are involved in chain length determination.Figure 2HPLC analysis of ubiquinone extracted from KO229 harboring various mutant ispB genes.Ubiquinone was extracted from E. coli KO229 harboring eachispB gene mutated as follows: A, wild-type (w. t.) IspB; B, L31V; C, I32V;D, Y38A; E, Y37A/Y38A; F, Y38A/R321V;G, Y61V; H, F75A; I, K235L;J, R321A; K, R321D; L, R321V.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although ispB in E. coli is essential for growth, the chromosomal COQ1 gene (homolog of ispB) inS. cerevisiae can be deleted to produce a respiration-deficient phenotype. We took advantage of thisCOQ1 mutant phenotype for analysis of the function ofispB (8Okada K. Suzuki K. Kamiya Y. Zhu X. Fujisaki S. Nishimura Y. Nishino T. Nakagawa T. Kawamukai M. Matsuda H. Biochim. Biophys. Acta. 1996; 1302: 217-223Crossref PubMed Scopus (104) Google Scholar). To express ispB mutants and to analyze their ubiquinone production in YKK6 (COQ1::URA3), mutant ispB genes fused with 53 amino acids of the Coq1 mitochondrial import signal were constructed. YKK6 was transformed with various mutant ispB fusion plasmids, and transformants were replicated on Synthetic Complete−Leu−Ura plates containing glucose (Fig. 3 A) or glycerol (Fig.3 B) as a non-fermentable carbon source. Although most of the strains grew on the glycerol plate, the Y37A, A79Y, K170G, and K170A mutant YKK6 strains did not grow, indicating that these mutants do not retain functional activity. These results are consistent with the complementation analysis of mutants in E. coli KO229/pKA3 (Fig. 2). We next analyzed the ubiquinone species produced by YKK6 harboring mutant ispB plasmids (Fig. 4). In the Y38A mutant, Q-8 was mainly produced, along with a significant amount of Q-7 (Fig. 4 C). In the Y37A/Y38A mutant, Q-8 and Q-7 were ma
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