Yeast and Rat Coq3 and Escherichia coli UbiG Polypeptides Catalyze Both O-Methyltransferase Steps in Coenzyme Q Biosynthesis
1999; Elsevier BV; Volume: 274; Issue: 31 Linguagem: Inglês
10.1074/jbc.274.31.21665
ISSN1083-351X
AutoresWayne W. Poon, Robert Barkovich, Adam Y. Hsu, Adam Frankel, Peter T. Lee, Jennifer Shepherd, David C. Myles, Catherine F. Clarke,
Tópico(s)Biochemical Acid Research Studies
ResumoUbiquinone (coenzyme Q or Q) is a lipid that functions in the electron transport chain in the inner mitochondrial membrane of eukaryotes and the plasma membrane of prokaryotes. Q-deficient mutants of Saccharomyces cerevisiae harbor defects in one of eight COQ genes (coq1–coq8) and are unable to grow on nonfermentable carbon sources. The biosynthesis of Q involves two separate O-methylation steps. In yeast, the first O-methylation utilizes 3,4-dihydroxy-5-hexaprenylbenzoic acid as a substrate and is thought to be catalyzed by Coq3p, a 32.7-kDa protein that is 40% identical to theEscherichia coli O-methyltransferase, UbiG. In this study, farnesylated analogs corresponding to the secondO-methylation step, demethyl-Q3 and Q3, have been chemically synthesized and used to study Q biosynthesis in yeast mitochondria in vitro. Both yeast and rat Coq3p recognize the demethyl-Q3 precursor as a substrate. In addition, E. coli UbiGp was purified and found to catalyze both O-methylation steps. Futhermore, antibodies to yeast Coq3p were used to determine that the Coq3 polypeptide is peripherally associated with the matrix-side of the inner membrane of yeast mitochondria. The results indicate that oneO-methyltransferase catalyzes both steps in Q biosynthesis in eukaryotes and prokaryotes and that Q biosynthesis is carried out within the matrix compartment of yeast mitochondria. Ubiquinone (coenzyme Q or Q) is a lipid that functions in the electron transport chain in the inner mitochondrial membrane of eukaryotes and the plasma membrane of prokaryotes. Q-deficient mutants of Saccharomyces cerevisiae harbor defects in one of eight COQ genes (coq1–coq8) and are unable to grow on nonfermentable carbon sources. The biosynthesis of Q involves two separate O-methylation steps. In yeast, the first O-methylation utilizes 3,4-dihydroxy-5-hexaprenylbenzoic acid as a substrate and is thought to be catalyzed by Coq3p, a 32.7-kDa protein that is 40% identical to theEscherichia coli O-methyltransferase, UbiG. In this study, farnesylated analogs corresponding to the secondO-methylation step, demethyl-Q3 and Q3, have been chemically synthesized and used to study Q biosynthesis in yeast mitochondria in vitro. Both yeast and rat Coq3p recognize the demethyl-Q3 precursor as a substrate. In addition, E. coli UbiGp was purified and found to catalyze both O-methylation steps. Futhermore, antibodies to yeast Coq3p were used to determine that the Coq3 polypeptide is peripherally associated with the matrix-side of the inner membrane of yeast mitochondria. The results indicate that oneO-methyltransferase catalyzes both steps in Q biosynthesis in eukaryotes and prokaryotes and that Q biosynthesis is carried out within the matrix compartment of yeast mitochondria. Ubiquinone is an essential lipid in the electron transport chain that is found in the inner mitochondrial membranes of eukaryotes and in the plasma membrane of prokaryotes (1Brandt U. Trumpower B. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 165-197Crossref PubMed Scopus (295) Google Scholar). The structure of Q 1The abbreviations used are: Q, ubiquinone or coenzyme Q; pHB, p-hydroxybenzoic acid; AdoMet, S-adenosyl-l-methionine; demethyl-Qn, demethyl-Q or 5-polyprenyl-2-hydroxy-3-methoxy-6-methyl-1,4-benzoquinone, wheren indicates the number of isoprenoids; COMT, catecholO-methyltransferase. consists of a quinone head group and a hydrophobic isoprenoid tail that can vary in length depending on the species in which it is found. The quinone group undergoes reversible single electron transfers, interchanging between the quinone, semiquinone, and hydroquinone, whereas the isoprenoid tail functions to anchor Q in the membrane. In eukaryotes, Q functions to shuttle electrons from either Complex I or Complex II to Complex III/bc1 complex. The transfer of electrons from Q to the bc1 complex is coupled to proton-translocation via the Q cycle mechanism that was first proposed by Mitchell (2Mitchell P. J. Theor. Biol. 1976; 62: 327-367Crossref PubMed Scopus (924) Google Scholar). A number of studies support such a mechanism (for a review, see Ref. 1Brandt U. Trumpower B. Crit. Rev. Biochem. Mol. Biol. 1994; 29: 165-197Crossref PubMed Scopus (295) Google Scholar) including the recently determined complete structure of the bc1 complex (3Iwata S. Lee J.W. Okada K. Lee J.K. Iwata M. Rasmussen B. Link T.A. Ramaswamy S. Jap B.K. Science. 1998; 281: 64-71Crossref PubMed Scopus (1068) Google Scholar). The redox properties of Q also allow it to function as a lipid soluble antioxidant. Q functions by either directly scavenging lipid peroxyl radicals (4Ernster L. Forsmark-Amdree P. Clin. Invest. 1993; 71: S60-S65Crossref PubMed Scopus (252) Google Scholar) or indirectly reducing α-tocopherol radicals to regenerate α-tocopherol (5Kagan V. Serbinova E. Packer L. Biochem. Biophys. Res. Commun. 1990; 169: 851-857Crossref PubMed Scopus (318) Google Scholar, 6Bowry V.W. Mohr D. Cleary J. Stocker R. J. Biol. Chem. 1995; 270: 5756-5763Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Additionally, Q protects cells from oxidative damage generated by the autoxidation of polyunsaturated fatty acids (7Do T. Schultz J.R. Clarke C.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7534-7539Crossref PubMed Scopus (143) Google Scholar). Q is found in many eukaryotic intracellular membranes, including the plasma membrane, where, in conjunction with a plasma membrane electron transport system, it functions to scavenge ascorbate free radicals (8Santos-Oscana C. Cordoba F. Crane F.L. Clarke C.F. Navas P. J. Biol. Chem. 1998; 273: 8099-8105Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 9Santos-Ocana C. Villalba J.M. Cordoba F. Padilla S. Crane F.L. Clarke C.F. Navas P. J. Bioenerg. Biomembr. 1998; 30: 465-475Crossref PubMed Scopus (55) Google Scholar). In the plasma membrane of prokaryotes, Q participates in the maintenance of the enzymatic activity of DsbA/DsbB disulfide bond forming proteins (10Kobayashi T. Kishigami S. Sone M. Inokuchi H. Mogi T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11857-11862Crossref PubMed Scopus (207) Google Scholar), and Q-deficient Escherichia coli strains are hypersensitive to thiol exposure (11Zeng H. Snavely I. Zamorano P. Javor G.T. J. Bacteriol. 1998; 180: 3681-3685Crossref PubMed Google Scholar). In both eukaryotes and prokaryotes, the first committed step in the biosynthesis of Q begins with the precursorsp-hydroxybenzoic acid (pHB) and isoprenoid diphosphate, in which the isoprenoid is covalently attached to the aromatic ring. The pathway derives from the characterization of accumulating Q biosynthetic intermediates in studies with Saccharomyces cerevisiae (12Olson R.E. Rudney H. Vitam. Horm. 1983; 40: 1-43Crossref PubMed Scopus (145) Google Scholar) and E. coli (13Gibson F. Biochem. Soc. Trans. 1973; 1: 317-326Crossref Google Scholar) Q-deficient mutants. In yeast, Q mutant strains have been classified into eight complementation groups, and five COQ genes have been characterized. The COQ1 and COQ2 genes encode the polyprenyl diphosphate synthase and the pHB:polyprenyldiphosphate transferase, respectively (14Ashby M.N. Edwards P.A. J. Biol. Chem. 1990; 265: 13157-13164Abstract Full Text PDF PubMed Google Scholar, 15Ashby M.N. Kutsunai S.Y. Ackerman S. Tzagoloff A. Edwards P.A. J. Biol. Chem. 1992; 267: 4128-4136Abstract Full Text PDF PubMed Google Scholar). The COQ3 gene encodes the O-methyltransferase thought to catalyze the firstO-methylation step (16Goewert R.R. Sippel J.C. Olson R.E. Biochemistry. 1981; 20: 4217-4223Crossref PubMed Scopus (21) Google Scholar, 17Clarke C.F. Williams W. Teruya J.H. J. Biol. Chem. 1991; 266: 16636-16644Abstract Full Text PDF PubMed Google Scholar), and the COQ5 gene encodes the C-methyltransferase in Q biosynthesis (18Barkovich R.J. Shtanko A. Shepherd J.A. Lee P.T. Myles D.C. Tzagoloff A. Clarke C.F. J. Biol. Chem. 1997; 272: 9182-9188Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 19Dibrov E. Robinson K.M. Lemire B.D. J. Biol. Chem. 1997; 272: 9175-9181Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Finally, the COQ7 gene encodes a protein that localizes to yeast mitochondria (20Jonassen T. Proft M. Randez-Gil F. Schultz J.R. Marbois B.N. Entian K.-D. Clarke C.F. J. Biol. Chem. 1998; 273: 3351-3357Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) and is required for the final monooxygenase step in Q biosynthesis (21Marbois B.N. Clarke C.F. J. Biol. Chem. 1996; 271: 2995-3004Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), but has also been implicated in aging and development in C. elegans (22Ewbank J.J. Barnes T.M. Lakowski B. Lussier M. Bussey H. Hekimi S. Science. 1997; 275: 980-983Crossref PubMed Scopus (264) Google Scholar). The Q biosynthetic pathway in E. coli has been carefully worked out by analyzing ubi mutant strains (23Gibson F. Young I.G. Methods Enzymol. 1978; 53: 600-609Crossref PubMed Scopus (26) Google Scholar) for accumulating Q intermediates at the blocked metabolic steps, and many of the bacterial genes have been characterized (24Meganathan G. Neidhardt F.C. Curtiss R. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. 2nd Ed. Esherichia coli and Salmonella, Cellular and Molecular Biology. 1. American Society for Microbiology, Washington D. C.1996: 642-656Google Scholar). These includeubiC, ispB, and ubiA, which encode the chorismate pyruvate lyase (25Siebert M. Severin K. Heide L. Microbiology. 1994; 140: 897-904Crossref PubMed Scopus (86) Google Scholar), octaprenyl synthase (26Okada K. Minehira M. Zhu X. Suzuki K. Nakagawa T. Matsuda H. Kawamukai M. J. Bacteriol. 1997; 179: 3058-3060Crossref PubMed Google Scholar), and the pHB:octaprenyltransferase (27Wu G. Williams H.D. Gibson F. Poole R.K. J. Gen. Microbiol. 1993; 139: 1795-1805Crossref PubMed Scopus (34) Google Scholar), respectively. Genes encoding the hydroxylase (ubiH) (28Nakahigashi K. Miyamoto K. Nishimura K. Inokuchi H. J. Bacteriol. 1992; 174: 7352-7359Crossref PubMed Google Scholar) and theO-methyltransferase (ubiG) (29Wu G. Williams H.D. Zamanian M. Gibson F. Poole R.K. J. Gen. Microbiol. 1992; 138: 2101-2112Crossref PubMed Scopus (66) Google Scholar, 30Hsu A.Y. Poon W.W. Shepherd J.A. Myles D.C. Clarke C.F. Biochemistry. 1996; 35: 9797-9806Crossref PubMed Scopus (97) Google Scholar) have also been reported, and recently, the gene encoding theC-methyltransferase gene in E. coli was characterized (ubiE) (31Lee P.T. Hsu A.Y. Ha H.T. Clarke C.F. J. Bacteriol. 1997; 179: 1748-1754Crossref PubMed Scopus (115) Google Scholar). Although eukaryotes and prokaryotes share many similar steps in Q biosynthesis, the pathway diverges after the prenylation step (16Goewert R.R. Sippel J.C. Olson R.E. Biochemistry. 1981; 20: 4217-4223Crossref PubMed Scopus (21) Google Scholar, 32Cox G.B. Young I.G. McCann L.M. Gibson F. J. Bacteriol. 1969; 99: 450-458Crossref PubMed Google Scholar, 33Goewert R.R. Sippel J.C. Grimm M.F. Olson R.E. Biochemistry. 1981; 20: 5611-5616Crossref PubMed Scopus (16) Google Scholar). In prokaryotes, decarboxylation, hydroxylation, and methylation follow prenylation, whereas in eukaryotes, the sequence is hydroxylation, methylation, and then decarboxylation. Recent evidence suggests that the Q biosynthetic pathway in higher eukaryotes is similar to S. cerevisiae. Both rat and human COQ3 and COQ7homologs can complement the corresponding defect in yeast (34Marbois B.N. Hsu A.Y. Pillai R. Colicelli J. Clarke C.F. Gene. 1994; 138: 213-217Crossref PubMed Scopus (40) Google Scholar, 35Jonassen T. Marbois B.N. Kim L. Chin A. Xia Y.-R. Lusis A.J. Clarke C.F. Arch. Biochem. Biophys. 1996; 330: 285-289Crossref PubMed Scopus (41) Google Scholar, 36Vajo Z. King L.M. Jonassen T. Wilkin D.J. Ho N. Minnich A. Clarke C.F. Francomano C.A. Mamm. Genome. 1999; (in press)PubMed Google Scholar). 2T. Jonassen and C. F. Clarke, unpublished data. We have been examining the enzymes that catalyze theO-methylations in prokaryotic and eukaryotic Q biosynthesis.E. coli strains harboring null mutations in theubiG gene are defective in the firstO-methylation step (conversion of compound 1 to2, Fig. 1) (30Hsu A.Y. Poon W.W. Shepherd J.A. Myles D.C. Clarke C.F. Biochemistry. 1996; 35: 9797-9806Crossref PubMed Scopus (97) Google Scholar). Surprisingly, strains harboring leaky mutant alleles of ubiG accumulate demethyl-Q8, the last intermediate in Q biosynthesis (Fig. 1, compound5), and are unable to carry out the lastO-methylation step (37Stroobant P. Young I.G. Gibson F. J. Bacteriol. 1972; 109: 134-139Crossref PubMed Google Scholar, 38Leppik R.A. Stroobant P. Shineberg B. Young I.G. Gibson F. Biochim. Biophys. Acta. 1976; 428: 146-156Crossref PubMed Scopus (32) Google Scholar). The analysis of both null and leaky mutant alleles of ubiG suggested that theubiG gene product was required for both of theO-methylations in Q biosynthesis (30Hsu A.Y. Poon W.W. Shepherd J.A. Myles D.C. Clarke C.F. Biochemistry. 1996; 35: 9797-9806Crossref PubMed Scopus (97) Google Scholar). Unlike the E. coli ubi mutants, analysis of accumulating Q intermediates in yeast coq mutants has been less informative. Yeast strains harboring coq3, coq4, coq5,coq6, coq7, or coq8 mutant alleles all accumulate the same single predominant intermediate, 3-hexaprenyl-4-hydroxybenzoic acid (39Poon W.W. Marbois B.N. Faull K.K. Clarke C.F. Arch. Biochem. Biophys. 1995; 320: 305-314Crossref PubMed Scopus (47) Google Scholar, 40Poon W.W. Do T.Q. Marbois B.N. Clarke C.F. Mol. Aspects Med. 1997; 18: s121-s127Crossref PubMed Scopus (58) Google Scholar). For this reason, it has often been instructive to compare the yeast COQ genes with the E. coli ubi gene counterparts. The encoded amino acid sequence of yeast COQ3 is 40% identical with the E. coli UbiG protein and both sequences contain the four motifs identified in a large family ofS-adenosyl-l-methionine (AdoMet)-dependent methyltransferases (41Kagan R.M. Clarke S. Arch. Biochem. Biophys. 1994; 310: 417-427Crossref PubMed Scopus (423) Google Scholar). In this study,in vitro assays have been developed that facilitate the study the catalytic role of both the UbiG and Coq3 proteins inO-methylation reactions. These assays demonstrate that each enzyme is active at all three O-methylation steps shown in Fig. 1. Mitochondria subfractionation studies indicate that the Coq3 polypeptide is a peripherally associated inner membrane protein, located on the matrix side. The results presented suggest that both the first and last O-methylation steps in the yeast Q biosynthetic pathway occur within the mitochondria matrix compartment. All reagents were used as received from Aldrich Chemical Co. unless otherwise noted. Unless specified as dry, the solvents were of unpurified reagent grade. Diethyl ether was distilled from sodium using benzophenone as an indicator. All air- or water-sensitive reactions were carried out under positive pressure of argon. Reactions were followed by TLC using Whatman precoated plates of silica gel 60 with fluorescent indicator. Reactions forming quinones were followed by leucomethylene blue stain. Normal phase flash chromatography was performed on Davisil Grade 643 silica gel (230–400 mesh). NMR spectra were measured on a Bruker ARX400 or ARX500 MHz spectrometer. Low and high resolution mass spectra were determined on a VG Autospec. Synthetic procedures used to generate farnesylated analogs of compounds 1, 2, 3, and 4 (Fig. 1) were described previously (30Hsu A.Y. Poon W.W. Shepherd J.A. Myles D.C. Clarke C.F. Biochemistry. 1996; 35: 9797-9806Crossref PubMed Scopus (97) Google Scholar,42.Shepherd, J. A., Synthesis of CoenzymeQ Biosynthetic Intermediates as Substrates for Q Methyltransferase in Vitro Assays.M.Sc. thesis, 1995, University of California, Los Angeles.Google Scholar, 43Shepherd J.A. Poon W.W. Myles D.C. Clarke C.F. Tetrahedron Lett. 1996; 37: 2395-2398Crossref Scopus (21) Google Scholar). In a glove bag under N2, AlCl3 (3.29 g, 24.7 mmol) was placed into a 100-ml round-bottomed flask. The flask was sealed and transferred to an argon atmosphere before anhydrous ether (15 ml) was slowly added, followed by 3,4,5-trimethoxytoluene (7) (2.8 ml, 16.6 mmol) and acetyl chloride (1.5 ml, 17.3 mmol). The reaction mixture turned dark and murky and was stirred for 20 h at room temperature. Following the addition of water (10 ml) and concentrated HCl (1 ml), the mixture was extracted with ether (three times, 15 ml). The combined ether layers were extracted with 1 m NaOH (three times, 20 ml), and the resulting aqueous layers were acidified by dropwise addition of concentrated HCl and then cooled in an ice-bath for 1 h. The product crystallized and was filtered using a Buchner funnel with Whatman No. 50 paper to give 1.56 g (44.6% yield) of pale yellow solid 8. 1H NMR (CDCl3, 400 MHz) δ 2.54 (s, 3H), 2.62 (s, 3H), 3.86 (s, 3H), 3.91 (s, 3H), 6.31 (s,3H), 11.97 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 204.33, 156.62, 155.87, 135.91, 134.46, 117.15, 106.90, 60.63, 55.84, 33.00, 24.37; LRMS m/z(relative intensity) EI 210.1 (72Neupert W. Annu. Rev. Biochem. 1997; 66: 863-917Crossref PubMed Scopus (978) Google Scholar), 195.1 (100), 180.0 (17Clarke C.F. Williams W. Teruya J.H. J. Biol. Chem. 1991; 266: 16636-16644Abstract Full Text PDF PubMed Google Scholar); HRMSm/z calculated for C11H14O4 (M+), 210.089067; found, 210.089209. Compound8 (180 mg, 0.86 mmol) was dissolved in a solution of sodium hydroxide (68 mg, 1.7 mmol) and water (4 ml). Hydrogen peroxide (0.12 ml, 30% in H2O) was added dropwise to the reaction mixture via an addition funnel over 10 min. The mixture was then heated at 45 °C for 2 h. Five minutes after the heating was initiated, the solution darkened from a pale yellow to a deep violet. The reaction was quenched by the addition of 1 m HCl (15 ml) and then extracted with dichloromethane (3 × 20 ml). The combined organic layers were dried over Na2SO4, filtered, and concentrated, by rotary evaporation. Flash chromatography using hexane:ethyl acetate (9:1) gave yellow solid 9 (80 mg, 51% yield). 1H NMR (CDCl3, 400 MHz) δ 3.22 (s,3H), 3.23 (s, 3H), 3.89 (s, 3H), 4.95 (s, 1H), 5.61 (s, 1H), 6.26 (s, 1H); 13C NMR (CDCl3, 100 MHz) 145.36, 136.39, 136.31, 134.06, 118.80, 105.41, 61.13, 56.25, 15.46; LRMS m/z (relative intensity) EI 184.1 (100), 169.0 (96), 154.1 (74Avelange-Macherel M.-H. Joyard J. Plant J. 1998; 14: 203-213Crossref PubMed Scopus (34) Google Scholar), 139.0 (20Jonassen T. Proft M. Randez-Gil F. Schultz J.R. Marbois B.N. Entian K.-D. Clarke C.F. J. Biol. Chem. 1998; 273: 3351-3357Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), 126.0 (25Siebert M. Severin K. Heide L. Microbiology. 1994; 140: 897-904Crossref PubMed Scopus (86) Google Scholar), 111.0 (83); HRMS m/z calculated for C9H12O4 (M+), 184.073485; found, 184.073559. Compound 9 (73.4 mg, 0.40 mmol) was dissolved in a 1:1 mixture of dichloromethane:acetonitrile (4 ml). A solution of ammonium cerium (IV) nitrate (655.4 mg, 1.20 mmol) in dichloromethane:acetonitrile (1:1, 2 ml) was then added dropwise to the reaction mixture over 5 min. The solution color changed from yellow to a turbid maroon. Stirring was continued for 5 min before the reaction was quenched by the addition of 10 ml of water. The reaction mixture was extracted with dichloromethane (three times, 15 ml) and the combined organic layers were concentrated by rotary evaporation. The crude residue was redissolved in ether (20 ml) and then treated with 1m NaHCO3 (20 ml). The aqueous layer became a bright violet color. Following two washes with ether, the aqueous layer was slowly acidified using concentrated HCl until the solution color changed from deep violet to orange. The aqueous layer was then extracted three times with ether. The combined ether extracts were dried over MgSO4, filtered, and concentrated by rotary evaporation to give fumigatin (56.5 mg, 84.4% yield), a red crystalline solid. 1H NMR (CDCl3, 400 MHz) δ 2.06 (d, J = 1.7 Hz, 3H), 4.09 (s, 3H), 6.39 (q, J = 1.7 Hz, 1H), 6.44 (br. s, 1H); 13C NMR (CDCl3, 100 MHz) δ 184.73, 183.45, 141.56, 140.02, 137.51, 132.44, 60.35, 14.93; LRMS m/z (relative intensity) EI 168.0 (100), 127.0 (48$$Google Scholar), 97.0 (33Goewert R.R. Sippel J.C. Grimm M.F. Olson R.E. Biochemistry. 1981; 20: 5611-5616Crossref PubMed Scopus (16) Google Scholar); HRMS m/z calculated for C8H8O4 (M+), 168.042536; found, 168.042259. The Freidel-Crafts allylation of 10 was performed as described (44Moore H.W. Folkers K. J. Am. Chem. Soc. 1966; 88: 564-567Crossref PubMed Scopus (5) Google Scholar) with the following modifications. Fumigatin (30.1 mg, 0.177 mmol) was dissolved in 1:1 ether:ethanol (6 ml), and then Na2S2O4 (10% in H2O) was added dropwise to the stirred solution until decolorization of the mixture was achieved. Ether (5 ml) was added to the decolorized mixture and the organic layer was washed three times with brine, dried over MgSO4, and concentrated in vacuo. The resulting hydroquinone of fumigatin was dissolved in freshly distilled 1, 4-dioxane (6 ml) under an argon atmosphere.Trans-trans-farnesol (118 mg, 0.53 mmol) was added to the solution, followed by BF3OEt2 (79.6 ml, 0.63 mmol), and the reaction was allowed to proceed for 18 h at room temperature. The reaction mixture was washed with brine and extracted three times with ether. The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The crude product was dissolved in ether (10 ml) and treated with excess FeCl3 in a 1:1 mixture of water:methanol for 30 min. The resulting mixture was extracted three times with ether, dried over MgSO4, filtered, and concentrated by rotary evaporation. The crude product was purified on a Florisil column with the following gradient system: 4:1 hexane/ethyl acetate, 1:1 hexane/ethyl acetate, 100% ethyl acetate, 4:1 hexane/ethyl acetate, 4:1 hexane/ethyl acetate with 1% glacial acetic acid. As described by Moore and Folkers (44Moore H.W. Folkers K. J. Am. Chem. Soc. 1966; 88: 564-567Crossref PubMed Scopus (5) Google Scholar), the desired product (demethyl-Q3) was retained as a bright purple compound at the top of the column until the final wash containing 1% glacial acetic acid was performed. Upon treatment with acetic acid, the color of the desired compound changed from purple to red-orange, and it was then eluted from the column. A yellow-orange oil (5) (18.5 mg, 28% yield) was obtained. 1H NMR (CDCl3, 500 MHz) δ 1.57 (s, 3H), 1.59 (s,3H), 1.67 (s, 3H), 1.74 (s, 3H), 1.98 (m, 8H), 2.04 (s, 3H), 3.20 (d, J = 7 Hz, 2H), 4.06 (s, 3H), 4.92 (t, J = 1 Hz, 1H), 5.06 (m, 2H), 6.48 (br. s, 1H); 13C NMR (CDCl3, 125 MHz) δ 185.14, 183.16, 143.36, 139.17, 137.82, 137.04, 136.16, 135.22, 131.30, 124.29, 123.81, 118.64, 60.26, 39.68, 26.74, 26.42, 35.68, 25.42, 17.65, 16.33, 16.01, 11.59; LRMS m/z (relative intensity) EI 372.2 (40Poon W.W. Do T.Q. Marbois B.N. Clarke C.F. Mol. Aspects Med. 1997; 18: s121-s127Crossref PubMed Scopus (58) Google Scholar), 267.0 (11Zeng H. Snavely I. Zamorano P. Javor G.T. J. Bacteriol. 1998; 180: 3681-3685Crossref PubMed Google Scholar), 236.1 (58Baker W. Raistrick H. J. Am. Chem. Soc. 1941; 1941: 670-672Crossref Scopus (8) Google Scholar), 221.1 (100), 183.1 (48$$Google Scholar), 162.0 (20Jonassen T. Proft M. Randez-Gil F. Schultz J.R. Marbois B.N. Entian K.-D. Clarke C.F. J. Biol. Chem. 1998; 273: 3351-3357Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), 121.1 (30Hsu A.Y. Poon W.W. Shepherd J.A. Myles D.C. Clarke C.F. Biochemistry. 1996; 35: 9797-9806Crossref PubMed Scopus (97) Google Scholar); HRMS m/z calculated for C23H32O4 (M+), 372.230403; found, 372.230060. The strains of S. cerevisiae used in the in vitro studies were JM45 (MATa, leu2–3, leu2–112, ura3–52, trp1–289, his4–580) (45McEwen J.E. Ko C. Kloeckner-Gruissem B. Poyton R.O. J. Biol. Chem. 1986; 261: 11872-11879Abstract Full Text PDF PubMed Google Scholar), a parent strain possessing Q synthesis, and JM45Δcoq3 (MATa,leu2–3, leu2-112, ura3–52, trp1–289, his4–580, coq3::LEU2) (17Clarke C.F. Williams W. Teruya J.H. J. Biol. Chem. 1991; 266: 16636-16644Abstract Full Text PDF PubMed Google Scholar). The yeast strains used in the localization studies were wild-type, W3031A (MATa, leu2–3, leu2–112, ura3–1, trp1–1, his3–11, ade2–1) (46Repetto B. Tzagoloff A. Mol. Cell. Biol. 1989; 9: 2695-2705Crossref PubMed Scopus (88) Google Scholar), and the Δcoq3 strain, CC3031B (MATα, leu2–3, leu2–112, ura3–1, trp1–1, his3–11, ade2–1, coq3Δ::LEU2) (7Do T. Schultz J.R. Clarke C.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7534-7539Crossref PubMed Scopus (143) Google Scholar). TheE. coli strain used was DH5α, which was obtained from Life Technologies, Inc. Growth media for yeast were prepared as described (47Adams A. Gottschling D.E. Kaiser C.A. Stearns T. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 145-149Google Scholar) and included YPD (1% yeast extract, 2% peptone, 2% dextrose), YPG (1% yeast extract, 2% peptone, 3% glycerol), YPGal (1% yeast extract, 2% peptone, 2% galactose) and SD-Ura (0.18% yeast nitrogen base without amino acids, 2% dextrose, 0.14% NaH2PO4, 0.5% (NH4)2SO4), with complete supplement minus uracil. The complete supplement was modified so that the final concentration of each component was as follows: 80 mg/liter, adenine sulfate, uracil, tryptophan, histidine, methionine, and cysteine; 40 mg/liter, arginine and tyrosine; 120 mg/liter, leucine; 60 mg/liter, isoleucine, lysine, and phenylalanine; 100 mg/liter, glutamic acid and aspartic acid; 150 mg/liter, valine; 200 mg/liter, threonine; and 400 mg/liter, serine). S. cerevisiae and E. coli were grown at 30 and 37 °C, respectively. DNA constructions were performed as described (48$$Google Scholar). pTHG was constructed to express UbiG as a fusion protein with a 33-amino acid N-terminal extension, containing 6 His residues (His6-UbiG) to provide for metal affinity column purification. A DNA segment containing the complete ubiG ORF (851–1572) was generated by a polymerase chain reaction with Vent DNA Polymerase (New England Biolabs) using pRPB (29Wu G. Williams H.D. Zamanian M. Gibson F. Poole R.K. J. Gen. Microbiol. 1992; 138: 2101-2112Crossref PubMed Scopus (66) Google Scholar) as the DNA template with the primers, pGB, (5′-GCGGATCCGATGAATGCCGAAAAATCGCCGGTA-3′) and pCC4K (30Hsu A.Y. Poon W.W. Shepherd J.A. Myles D.C. Clarke C.F. Biochemistry. 1996; 35: 9797-9806Crossref PubMed Scopus (97) Google Scholar). The resulting 723-base pair product was inserted after digestion withBamHI and KpnI into the similarly digested vector pTrcHisB (Invitrogen) to generate pTHG. The plasmids, pQM and pCHQ3, were described previously (30Hsu A.Y. Poon W.W. Shepherd J.A. Myles D.C. Clarke C.F. Biochemistry. 1996; 35: 9797-9806Crossref PubMed Scopus (97) Google Scholar). Purification of His6 UbiG was done with the TALON metal affinity resin (CLONTECH) as described by the manufacturer. The E. coli strain, DH5α:pTHG, containing the His6-UbiG was grown in LB+Amp (50 μg/ml) and induced with isopropyl-1-thio-β-d-galactopyranoside (final concentration, 0.4 mm), and cells were disrupted by the French press method. His6-UbiG was purified on a TALON column under native conditions. The resin was washed with 15 mm imidazole to remove nonspecifically bound proteins, His6-UbiG was eluted from the resin with 250 mmimidazole, and the imidazole was removed by dialysis against 0.05m sodium phosphate, pH 7.0. A plasmid encoding a glutathione S-transferase-Coq3p fusion protein was constructed by subcloning the 1.7-kilobase EcoRI fragment of pRS12A (17Clarke C.F. Williams W. Teruya J.H. J. Biol. Chem. 1991; 266: 16636-16644Abstract Full Text PDF PubMed Google Scholar) into the EcoRI site of pGEX-2T (Amersham Pharmacia Biotech). The fusion protein contained amino acids 64–316 of yeast Coq3p as a C-terminal fusion to glutathioneS-transferase and was produced in E. coli and the insoluble fraction was separated by preparative SDS-polyacrylamide gel electrophoresis. The 50-kDa fusion protein was visualized by copper staining (49Garfin D.E. Methods Enzymol. 1990; 182: 425-441Crossref PubMed Scopus (284) Google Scholar) and eluted from the gel by diffusion (50Harrington M.G. Methods Enzymol. 1990; 182: 488-495Crossref PubMed Scopus (50) Google Scholar). The protein was injected into rabbits, and antibodies were affinity purified according to standard techniques (51Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). Assays for O-methyltransferase activities were determined with the three synthetic methyl-acceptors, compounds 1, 3, and 5. Stocks of1, 3, and 5 were stored undiluted at −20 °C under argon. In assays with either 1 or3, the substrates were redissolved into methanol, and each reaction mixture (250 μl) contained 0.05 m sodium phosphate, pH 7.0, 1.0 mm ZnSO4, and 50 μl of crude yeast mitochondria (0.25–0.50 mg of protein) (52Glick P.S. Pon L.A. Methods Enzymol. 1995; 260: 213-233Crossref PubMed Scopus (287) Google Scholar) or purifiedE. coli protein, His6-UbiG (1.2 ng). The final concentration of compound 1 or 3 in each assay was 50 μm unless otherwise stated. Reactions were started with the addition ofS-adenosyl-l-[methyl- 3H]methionine to a final concentration of 60 μm (NEN Life Science Products, 84.1 Ci/mmol; specific activity was adjusted to 560 mCi/mmol with unlabeled S-adenosyl-l-methionine). The concentration of S-adenosyl-l-methionine was determined by its absorbance at 256 nm (ε 15, 200m−1 cm−1) (53Dawson R.M.C. Elliott D.C. Elliott W.H. Jones K.M. Data for Biochemical Research. 3rd Ed. Oxford University Press, New York1986: 2-3Google Scholar). After incubation, the reaction was stopped by
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