Isolation and Characterization of the Versicolorin B Synthase Gene from Aspergillus parasiticus
1996; Elsevier BV; Volume: 271; Issue: 23 Linguagem: Inglês
10.1074/jbc.271.23.13600
ISSN1083-351X
AutoresJeffrey C. Silva, Robert E. Minto, Clifford E. Barry, Koren A. Holland, Craig A. Townsend,
Tópico(s)Fungal and yeast genetics research
ResumoVersicolorin B synthase catalyzes the side chain cyclization of racemic versiconal hemiacetal (7Loechler E.L. Teeter M.M. Whitlow M.D. J. Biomol. Struct. & Dyn. 1988; 5: 1237-1257Crossref PubMed Scopus (23) Google Scholar) to the bisfuran ring system of (−)-versicolorin B (8Neal G.E. Nature. 1973; 244: 432-435Crossref PubMed Scopus (36) Google Scholar), an essential transformation in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. The dihydrobisfuran is key to the mutagenic nature of aflatoxin B1 (1Lloyd A.T. Sharp P.M. Mol. & Gen. Genet. 1991; 230: 288-294Crossref PubMed Scopus (60) Google Scholar). The protein, which shows 58% similarity and 38% identity with glucose oxidase from Aspergillus niger, possesses an amino-terminal sequence homologous to the ADP-binding region of other flavoenzymes. However, this enzyme does not require flavin or nicotinamide cofactors for its cyclase activity. The 643-amino acid native enzyme contains three potential sites for N-linked glycosylation, Asn-Xaa-Thr or Asn-Xaa-Ser. The cDNA and genomic clones of versicolorin B synthase were isolated by screening the respective libraries with random-primed DNA probes generated from an exact copy of an internal vbs sequence. This probe was created through polymerase chain reaction by using nondegenerate polymerase chain reaction primers derived from the amino acid sequences of peptide fragments of the enzyme. The 1985-base genomic vbs DNA sequence is interrupted by one intron of 53 nucleotides. Southern blotting, nucleotide sequencing, and detailed restriction mapping of the vbs-containing genomic clones revealed the presence of omtA, a methyltransferase active in the biosynthesis, 3.3 kilobases upstream of vbs and oriented in the opposite direction from vbs. The presence of omtA in close proximity to vbs supports the theory that the genes encoding the aflatoxin biosynthetic enzymes in A. parasiticus are clustered. Versicolorin B synthase catalyzes the side chain cyclization of racemic versiconal hemiacetal (7Loechler E.L. Teeter M.M. Whitlow M.D. J. Biomol. Struct. & Dyn. 1988; 5: 1237-1257Crossref PubMed Scopus (23) Google Scholar) to the bisfuran ring system of (−)-versicolorin B (8Neal G.E. Nature. 1973; 244: 432-435Crossref PubMed Scopus (36) Google Scholar), an essential transformation in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. The dihydrobisfuran is key to the mutagenic nature of aflatoxin B1 (1Lloyd A.T. Sharp P.M. Mol. & Gen. Genet. 1991; 230: 288-294Crossref PubMed Scopus (60) Google Scholar). The protein, which shows 58% similarity and 38% identity with glucose oxidase from Aspergillus niger, possesses an amino-terminal sequence homologous to the ADP-binding region of other flavoenzymes. However, this enzyme does not require flavin or nicotinamide cofactors for its cyclase activity. The 643-amino acid native enzyme contains three potential sites for N-linked glycosylation, Asn-Xaa-Thr or Asn-Xaa-Ser. The cDNA and genomic clones of versicolorin B synthase were isolated by screening the respective libraries with random-primed DNA probes generated from an exact copy of an internal vbs sequence. This probe was created through polymerase chain reaction by using nondegenerate polymerase chain reaction primers derived from the amino acid sequences of peptide fragments of the enzyme. The 1985-base genomic vbs DNA sequence is interrupted by one intron of 53 nucleotides. Southern blotting, nucleotide sequencing, and detailed restriction mapping of the vbs-containing genomic clones revealed the presence of omtA, a methyltransferase active in the biosynthesis, 3.3 kilobases upstream of vbs and oriented in the opposite direction from vbs. The presence of omtA in close proximity to vbs supports the theory that the genes encoding the aflatoxin biosynthetic enzymes in A. parasiticus are clustered. INTRODUCTIONAflatoxin B1 (see Scheme I, 1), the principal member of the aflatoxin family, is one of the most potent mycotoxins known to man. The imperfect fungi Aspergillus parasiticus, Aspergillus flavus, and Aspergillus nomius produce aflatoxins, and these fungi are known to infect corn, grains, and nuts during their growth and during storage leading to the introduction of aflatoxin into primary foodstuffs (2Lillehoj E.B. Hesseltine C.W. Rodricks J.V. Hesseltine C.W. Mehlman M.A. Mycotoxins in Human and Animal Health. Pathotox Publishers, Park Forest South, IL1977: 107Google Scholar, 3Dickens J.W. Rodricks J.V. Hesseltine C.W. Mehlman M.A. Mycotoxins in Human and Animal Health. Pathotox Publishers, Park Forest South, IL1977: 99Google Scholar). The natural product AFB1 1The abbreviations used are: AFB1aflatoxin B1MeCNacetonitrileVBSversicolorin B synthasePCRpolymerase chain reactionkbkilobase(s)bpbase pair(s)HPLChigh pressure liquid chromatographygDNAgenomic DNA. itself does not pose a major health threat; however, renal and hepatic oxidative detoxification of AFB1-contaminated foods by P450 enzymes yields aflatoxin-15,16-exo-epoxide (see Scheme I, 2), a highly toxic mutagen (4Baertschi S.W. Raney K.D. Stone M.P. Harris T.M. J. Am. Chem. Soc. 1988; 110: 7923-7931Crossref Scopus (240) Google Scholar, 5Martin C.N. Garner R.C. Nature. 1977; 267: 863-865Crossref PubMed Scopus (163) Google Scholar). It has been shown that the epoxide targets guanine residues and selectively alkylates the N-7 position of this purine in double-stranded DNA (6Essigmann J.M. Croy R.G. Nadzan A.M. Busby Jr., W.F. Reinhold V.N. Büchi G. Wogan G.N. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 1874-1970Crossref Scopus (522) Google Scholar, 7Loechler E.L. Teeter M.M. Whitlow M.D. J. Biomol. Struct. & Dyn. 1988; 5: 1237-1257Crossref PubMed Scopus (23) Google Scholar). Depurination of the alkylated base has been correlated to bladder cancer in laboratory mice (8Neal G.E. Nature. 1973; 244: 432-435Crossref PubMed Scopus (36) Google Scholar, 9Saunders F.C. Barker E.A. Smuckler E.A. Cancer Res. 1972; 32: 2487-2494PubMed Google Scholar, 10Gelboin H.V. Wortham J.S. Wilson R.G. Friedman M. Wogan G.N. Science. 1966; 154: 1205-1206Crossref PubMed Scopus (54) Google Scholar), teratogenic effects in chicken embryos (11Asplin F.D. Carnaghan R.B.A. Vet. Rec. 1961; 73: 1215-1219Google Scholar), and liver cancer in humans (12Campbell T.C. Chen J. Liu C. Li J. Parpia B. Cancer Res. 1990; 50: 6882-6893PubMed Google Scholar, 13Peers F.G. Gilman G.A. Linsell C.A. Int. J. Cancer. 1976; 17: 167-176Crossref PubMed Scopus (153) Google Scholar, 14Van-Rensburg S.J. Schalkwyk G.C. Schalkwyk D.J. J. Environ. Pathol. Toxicol. Oncol. 1990; 10: 11-16PubMed Google Scholar). A direct connection between DNA damage and the incidence of human cancer has been established to originate at mutational hot spots of the p53 gene, a tumor suppressor gene whose altered sequence has been associated with approximately 50% of all human cancers (15Hollstein M. Sidransky D. Vogelstein B. Harris C.C. Science. 1991; 253: 49-53Crossref PubMed Scopus (7411) Google Scholar, 16Eaton D.L. Gallagher E.P. Annu. Rev. Pharmacol. Toxicol. 1994; 34: 135-172Crossref PubMed Scopus (684) Google Scholar). Aflatoxin B1 has been found to be responsible in particular for G → T transversions at codon 249 of the p53 tumor suppressor gene in hepatocarcinogenesis (17Hsu I.C. Metcalf R.A. Sun T. Wesh J.A. Wang N.J. Harris C.C. Nature. 1991; 350: 427-428Crossref PubMed Scopus (1406) Google Scholar, 18Aguilar F. Hussain S.P. Cerutti P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8586-8590Crossref PubMed Scopus (410) Google Scholar) (Scheme I).The aflatoxin biosynthetic pathway is notably long and complex (Scheme II). Although the formation of polyketide natural products is initiated normally by acetate, a specialized fatty acid synthase apparently acts in the case of aflatoxin to generate a six-carbon hexanoyl starter unit. This primer is homologated by successive malonyl condensations to give, after intramolecular aldol condensation, cyclization, and oxidation, norsolorinic acid (3) (19Brobst S.W. Townsend C.A. Can. J. Chem. 1993; 72: 200-207Crossref Scopus (41) Google Scholar, 20Lee L.S. Bennett J.W. Goldblatt L.A. Lundin R.E. J. Am. Oil Chem. Soc. 1971; 48: 93-94Crossref PubMed Scopus (48) Google Scholar, 21Townsend C.A. Brobst S.W. Ramer S.E. Vederas J.C. J. Am. Chem. Soc. 1988; 110: 318-319Crossref Scopus (20) Google Scholar). Simple redox changes in the hexanoyl side chain yield the internal ketal averufin (4) (22Hsieh D.P.H. Lin M.T. Yao R.C. Singh R. J. Agric. Food Chem. 1976; 24: 1170-1174Crossref PubMed Scopus (42) Google Scholar, 23Yabe K. Nakamura Y. Nakajima H. Ando Y. Hamasaki T. Appl. Environ. Microbiol. 1991; 57: 1340-1345Crossref PubMed Google Scholar, 24Yabe K. Matsuyama Y. Ando Y. Nakajima H. Hamasaki T. Appl. Environ. Microbiol. 1993; 59: 2486-2492Crossref PubMed Google Scholar). Oxidation at C-2′ of 4 induces migration of the anthraquinone nucleus from C-1′ to C-2′ to afford hydroxyversicolorone (5) containing the first furan ring (25Townsend C.A. Plavcan K.A. Pal K. Brobst S.W. Irish M.S. Ely Jr., E.W. Bennett J.W. J. Org. Chem. 1988; 53: 2472-2477Crossref Scopus (21) Google Scholar, 26Townsend C.A. Whittamore P.R.O. Brobst S.W. J. Chem. Soc. Chem. Commun. 1988; : 726-728Crossref Google Scholar). Preparatory to formation of the second furan ring, oxygen is inserted into the carbon chain of 5 by a proposed Baeyer Villiger-like reaction to give versiconal acetate (6) (27Townsend C.A. Christensen S.B. Davis S.G. J. Am. Chem. Soc. 1982; 104: 6154-6155Crossref Scopus (46) Google Scholar, 28McGuire S.M. Townsend C.A. Bioorg. & Med. Chem. Lett. 1993; 3: 653-656Crossref Scopus (22) Google Scholar). Support for this mechanism has come from a fermentation conducted in an 18O2-containing atmosphere in which the isotopic label (*) was specifically incorporated at the ester oxygen (Scheme II) as shown in 6 (28McGuire S.M. Townsend C.A. Bioorg. & Med. Chem. Lett. 1993; 3: 653-656Crossref Scopus (22) Google Scholar). A cell-free system of A. parasiticus has been described in which an esterase catalyzed the hydrolysis of this terminal acetate to give versiconal (7), which was cyclized to (−)-versicolorin B (8) (29McGuire S.M. Brobst S.W. Graybill T.L. Pal K. Townsend C.A. J. Am. Chem. Soc. 1989; 111: 8308-8309Crossref Scopus (25) Google Scholar). Tracing the fate of 18O label (*) from 6, it was shown that heavy isotope was retained in 7 without loss in the critical cyclization to versicolorin B (8) (27Townsend C.A. Christensen S.B. Davis S.G. J. Am. Chem. Soc. 1982; 104: 6154-6155Crossref Scopus (46) Google Scholar, 28McGuire S.M. Townsend C.A. Bioorg. & Med. Chem. Lett. 1993; 3: 653-656Crossref Scopus (22) Google Scholar). In hemiacetals 5, 6, and 7, the chiral C-2′ center is benzylic and adjacent to a masked aldehyde. This is an intrinsically labile stereocenter, and each of these three compounds is isolated as a racemate (25Townsend C.A. Plavcan K.A. Pal K. Brobst S.W. Irish M.S. Ely Jr., E.W. Bennett J.W. J. Org. Chem. 1988; 53: 2472-2477Crossref Scopus (21) Google Scholar, 30Steyn P.S. Vleggar R. Wessels P.L. Cole R.J. Scott D.B. J. Chem. Soc. Perkin Trans. I. 1979; 1: 451-459Crossref Scopus (28) Google Scholar, 31Townsend C.A. Isomura Y. Davis S.G. Hodge J.A. Tetrahedron. 1989; 45: 2263-2276Crossref Scopus (23) Google Scholar) (Scheme II).Scheme IIAflatoxin B1 biosynthetic pathway.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The cyclization of versiconal hemiacetal (7) can be carried out nonenzymatically in the presence of acid to yield versicolorin B (8) as its racemate, which is designated historically as versicolorin C (32Hatsuda Y. Hamasaki T. Ishida M. Kiyama Y. Agric. Biol. Chem. 1971; 35: 444Google Scholar, 33Cole R.J. Cox R.H. Handbook of Toxic Fungal Metabolites. Academic Press, New York1981: 1Crossref Google Scholar). At neutral pH this chemical process is slow and cyclization is catalyzed in vivo by versicolorin B synthase (VBS) to give optically active (−)-versicolorin B (8) (29McGuire S.M. Brobst S.W. Graybill T.L. Pal K. Townsend C.A. J. Am. Chem. Soc. 1989; 111: 8308-8309Crossref Scopus (25) Google Scholar, 30Steyn P.S. Vleggar R. Wessels P.L. Cole R.J. Scott D.B. J. Chem. Soc. Perkin Trans. I. 1979; 1: 451-459Crossref Scopus (28) Google Scholar, 31Townsend C.A. Isomura Y. Davis S.G. Hodge J.A. Tetrahedron. 1989; 45: 2263-2276Crossref Scopus (23) Google Scholar, 32Hatsuda Y. Hamasaki T. Ishida M. Kiyama Y. Agric. Biol. Chem. 1971; 35: 444Google Scholar, 33Cole R.J. Cox R.H. Handbook of Toxic Fungal Metabolites. Academic Press, New York1981: 1Crossref Google Scholar). 2S. M. McGuire, J. C. Silva, E. G. Casillas, and C. A. Townsend, manuscript submitted. The absolute configuration installed in this cyclase-catalyzed step is preserved in the bisfuran throughout the remainder of the biosynthetic pathway to AFB1. The stereochemical match of this structure when metabolically activated as the exo-epoxide 2 and intercalated into right-handed helical DNA is essential to successful covalent adduct formation (35Gopalakrishnan S. Harris T.M. Stone M.P. Biochemistry. 1990; 29: 10438-10448Crossref PubMed Scopus (87) Google Scholar). These are key events in the tumorgenesis of this natural product. Preliminary purifications of VBS have been reported (36Anderson J.A. Green L.D. Mycopathologia. 1994; 126: 169-172Crossref Scopus (3) Google Scholar, 37Anderson J.A. Chung C.H. Mycopathologia. 1990; 110: 31-35Crossref PubMed Scopus (13) Google Scholar, 38Townsend C.A. McGuire S.M. Brobst S.W. Graybill T.L. Pal K. Barry III, C.E. Rosenkranz H.S. Secondary Metabolite Biosynthesis and Metabolism. Plenum Publishing Corp., New York1992: 141Google Scholar), but an improved protocol yielding homogeneous protein has been achieved.2 Detailed kinetic analyses of the reaction catalyzed by this enzyme reveal that from the stereochemically equilibrating mixture of enantiomers of 7, the 2′S-configured hemiacetal is specifically cyclized by VBS to (−)-versicolorin B (8).2Formation of the dihydrobisfuran is completed in the oxidative desaturation of versicolorin B (8) to versicolorin A (9) (29McGuire S.M. Brobst S.W. Graybill T.L. Pal K. Townsend C.A. J. Am. Chem. Soc. 1989; 111: 8308-8309Crossref Scopus (25) Google Scholar, 39Yabe K. Ando Y. Hamasaki T. Agric. Biol. Chem. 1991; 55: 1907-1911Google Scholar). The subsequent steps of the biosynthetic pathway are significantly less well understood. Cleavage of the anthraquinone nucleus and cyclization, decarboxylation, and dehydration afford the xanthone 10 (40Hsieh D.P.H. Lin M.T. Yao R.C. Biochem. Biophys. Res. Commun. 1973; 52: 992-997Crossref PubMed Scopus (57) Google Scholar). Successive O-methylations are known to occur at C-5 and C-7 to give O-methylsterigmatocystin (11) (41Cleveland T.E. Lax A.R. Lee L.S. Bhatnagar D. Appl. Environ. Microbiol. 1987; 53: 1711-1713Crossref PubMed Google Scholar, 42Bhatnagar D. Cleveland T.E. Biochimie (Paris). 1988; 70: 743-747Crossref PubMed Scopus (9) Google Scholar, 43Yabe K. Ando Y. Hashimoto J. Hamasaki T. Appl. Environ. Microbiol. 1989; 55: 2172-2177Crossref PubMed Google Scholar, 44Keller N.P. Dischinger H.C. Bhatnagar D. Cleveland T.E. Ullah A.H.J. Appl. Environ. Microbiol. 1993; 59: 479-484Crossref PubMed Google Scholar). This intermediate is further cleaved oxidatively, demethylated, cyclized, and decarboxylated to ultimately afford aflatoxin B1 (1) (45Watanabe C.M.H. Townsend C.A. J. Org. Chem. 1996; 61: 1990-1993Crossref Scopus (26) Google Scholar, 46Chatterjee M. Townsend C.A. J. Org. Chem. 1994; 59: 4424-4429Crossref Scopus (21) Google Scholar, 47Cleveland T.E. Bhatnagar D. Can. J. Microbiol. 1987; 33: 1108-1112Crossref PubMed Scopus (22) Google Scholar).Although the mechanisms of these deep-seated molecular rearrangements in the post-versicolorin A segment of the pathway are not known, important progress has been made recently to identify the first genes in A. parasiticus that encode proteins involved in the biosynthesis of aflatoxin (48Skory G.D. Chang P.K. Cary J. Linz J.E. Appl. Environ. Microbiol. 1992; 58: 3527-3537Crossref PubMed Google Scholar, 49Trail F. Chang P.-K. Cary J. Linz J.E. Appl. Environ. Microbiol. 1994; 60: 4078-4085Crossref PubMed Google Scholar, 50Chang P.K. Cary J.W. Bhatnagar D. Cleveland T.E. Bennett J.W. Linz J.E. Woloshuk C.P. Payne G.A. Appl. Environ. Microbiol. 1993; 59: 3273-3279Crossref PubMed Google Scholar, 51Yu J. Chang P.-K. Cary J.W. Wright M. Bhatnagar D. Cleveland T.E. Payne G.A. Linz J.E. Appl. Environ. Microbiol. 1995; 61: 2365-2371Crossref PubMed Google Scholar). Preliminary evidence has been gathered to suggest that these genes are substantially clustered (48Skory G.D. Chang P.K. Cary J. Linz J.E. Appl. Environ. Microbiol. 1992; 58: 3527-3537Crossref PubMed Google Scholar, 49Trail F. Chang P.-K. Cary J. Linz J.E. Appl. Environ. Microbiol. 1994; 60: 4078-4085Crossref PubMed Google Scholar, 50Chang P.K. Cary J.W. Bhatnagar D. Cleveland T.E. Bennett J.W. Linz J.E. Woloshuk C.P. Payne G.A. Appl. Environ. Microbiol. 1993; 59: 3273-3279Crossref PubMed Google Scholar, 51Yu J. Chang P.-K. Cary J.W. Wright M. Bhatnagar D. Cleveland T.E. Payne G.A. Linz J.E. Appl. Environ. Microbiol. 1995; 61: 2365-2371Crossref PubMed Google Scholar), contrary to earlier reports (52Bennett J.W. Vinnett C.H. Goynes W.R.J. Can. J. Microbiol. 1980; 26: 706-713Crossref PubMed Scopus (11) Google Scholar, 53Papa K.E. Mycologia. 1978; 70: 766-773Crossref PubMed Google Scholar, 54Papa K.E. J. Gen. Microbiol. 1982; 128: 1345-1348Google Scholar, 55Papa K.E. Can. J. Microbiol. 1984; 30: 68-73Crossref PubMed Scopus (25) Google Scholar). A probable polyketide synthase (pksA) and two fatty acid synthase (fas-1A and fas-2A) genes have been identified by sequence homology and gene disruption experiments (51Yu J. Chang P.-K. Cary J.W. Wright M. Bhatnagar D. Cleveland T.E. Payne G.A. Linz J.E. Appl. Environ. Microbiol. 1995; 61: 2365-2371Crossref PubMed Google Scholar). The localization of two genes, a ketoreductase (nor-1) acting immediately after the formation of norsolorinic acid (3) and ver-1, whose gene product participates in the oxidative cleavage of versicolorin A (9), has been determined by gene disruption and complementation (48Skory G.D. Chang P.K. Cary J. Linz J.E. Appl. Environ. Microbiol. 1992; 58: 3527-3537Crossref PubMed Google Scholar, 49Trail F. Chang P.-K. Cary J. Linz J.E. Appl. Environ. Microbiol. 1994; 60: 4078-4085Crossref PubMed Google Scholar, 56Skory C.D. Chang P.-K. Linz J.E. Appl. Environ. Microbiol. 1993; 59: 1642-1646Crossref PubMed Google Scholar). Combined with the cloning of one of the purified O-methyltransferases (omtA), the direct linkage of these genes has been determined to be within 45 kb of one another (see Fig. 5) (51Yu J. Chang P.-K. Cary J.W. Wright M. Bhatnagar D. Cleveland T.E. Payne G.A. Linz J.E. Appl. Environ. Microbiol. 1995; 61: 2365-2371Crossref PubMed Google Scholar). In this paper we describe the isolation of the gene encoding versicolorin B synthase (vbs) from both cDNA and gDNA libraries derived from A. parasiticus. Comparison of the sequences reveals the presence of a single intron in the latter. Translation of the mature mRNA gives a protein of 70,226 Da, in modest agreement with the 78-kDa apparent molecular mass of wild-type VBS as judged by its relative electrophoretic mobility.2 Alignment of the translated amino acid sequence of VBS with protein sequences compiled in protein data bases revealed a marked homology to several flavin-dependent oxidases and dehydrogenases. This relationship was unexpected because VBS does not catalyze a redox reaction. Finally, mapping of vbs gDNA clones has allowed the locus of this gene to be established about 3.3 kb upstream of omtA and separated from it by an apparent cytochrome P450 monooxygenase 3R. E. Minto and C. A. Townsend, unpublished results. approximately 1400 bp in length of unknown function. These findings expand the experimentally determined dimensions of the apparent aflatoxin gene cluster and unambiguously define the function and location of the gene encoding versicolorin B synthase.Fig. 5Further characterization of the A. parasiticus partial gene cluster for the aflatoxin B1 biosynthetic pathway. a, recently published gene cluster of AFB1 biosynthetic genes of approximately 60 kb. b, lambda clone λ62b, approximately 15 kb, extending the existing AFB1 gene cluster to include versicolorin B synthase (vbs), and an apparent cytochrome P450 by amino acid homology of the translated mRNA sequence. c, lambda clone λ52a, approximately 18 kb, where vbs is truncated at the 5′ end (·). The sizes of the EcoRI restriction fragments are indicated in bold above the mapped DNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)RESULTSVBS, a homodimeric protein of 78-kDa subunits, catalyzes the dehydrative cyclization of racemic versiconal hemiacetal (7) to optically active versicolorin B (8), the step penultimate to desaturation of the tetrahydrobisfuran to the dihydrobisfuran present in (−)-versicolorin A (9) (29McGuire S.M. Brobst S.W. Graybill T.L. Pal K. Townsend C.A. J. Am. Chem. Soc. 1989; 111: 8308-8309Crossref Scopus (25) Google Scholar, 30Steyn P.S. Vleggar R. Wessels P.L. Cole R.J. Scott D.B. J. Chem. Soc. Perkin Trans. I. 1979; 1: 451-459Crossref Scopus (28) Google Scholar, 31Townsend C.A. Isomura Y. Davis S.G. Hodge J.A. Tetrahedron. 1989; 45: 2263-2276Crossref Scopus (23) Google Scholar, 32Hatsuda Y. Hamasaki T. Ishida M. Kiyama Y. Agric. Biol. Chem. 1971; 35: 444Google Scholar, 33Cole R.J. Cox R.H. Handbook of Toxic Fungal Metabolites. Academic Press, New York1981: 1Crossref Google Scholar).2 This unique structural feature is conserved through the subsequent intermediates of aflatoxin B1 (1) biosynthesis (Scheme II) and is the seat of the progressively severe carcinogenic properties of these metabolites. VBS was purified to homogeneity from A. parasiticus SU-1 (ATCC 56775) by methods established in this laboratory,2 but failed to give amino-terminal sequence data by automated methods. Although homogenous enzyme was submitted for amino acid sequence analysis (250 pmol), amino acid intensities corresponding to ≤10 pmol of enzyme were observed, suggesting that the amino terminus of the native protein was post-translationally modified. To circumvent this problem, the protein was digested with Lys-C endopeptidase and two of approximately 20 VBS peptide fragments were isolated by reverse-phase HPLC. These two peptide fragments both gave reproducible amino acid sequence data and credible stoichiometry (Fig. 1).The two peptide sequences were used to design seven oligonucleotide probes for hybridization and PCR experiments. The degeneracy of these probes was minimized by comparing codon usage in A. nidulans and A. niger structural genes to compile a table of codon biases.4 Plaque hybridization experiments did not provide reproducible results using the partially degenerate probes 7NC, 8NC, 9NC, 10C, 13C, 14C, and 15C synthesized by automated methods (Fig. 1). However, PCR-generated nondegenerate probes were later substituted for these degenerate probes in the hybridization experiments, as described under "Experimental Procedures," to lead to the successful cloning of VBS.A vbs cDNA gene fragment, obtained from the reverse transcriptase-mediated PCR, was amplified using PCR primers 8NC and 10C and estimated to be approximately 750 bp in length. No PCR product was obtained using any other primer combination from the set of primers shown in Fig. 1. PCR amplification of the approximately 750-bp fragment was observed using both 48- and 60-h mRNA. This gene fragment was subcloned into pBluescriptII SK(−) generating clones with the insert oriented in both directions (pRPF1-17C and pRPF1-13NC). Single-stranded DNA was prepared by infecting the plasmid-borne XL1-Blue cells with R408 helper phage. Direct sequencing was performed using dideoxy sequencing methods in both directions using clones containing inserts in opposite orientations. Partial nucleotide sequences from the coding (pRPF1-17C) and the noncoding strands (pRPF1-13NC) can be seen in Fig. 4. Translation of the two nucleotide sequences of both pRPF1-17C and pRPF1-13NC concurred with the amino acid sequence data obtained from each of the two peptide fragments isolated from the Lys-C endopeptidase treatment of VBS (Fig. 4, pRPF1-13NC, and pRPF1-17C nucleotide sequences).From these nucleotide sequences, nondegenerate primers 21C and 22NC were prepared to serve as oligonucleotide primers for PCR experiments with both the gDNA and cDNA libraries (Fig. 4). A 615-bp internal fragment was successfully amplified by PCR from both the cDNA and gDNA libraries. This PCR fragment was used to generate oligonucleotide probes for plaque hybridizations as described under "Experimental Procedures." Further PCR analysis of the gDNA and cDNA clones with primers 8NC and 10C afforded two discrete gene products from each set of clones approximately 800 and 750 bp in length, respectively. The approximately 50-bp difference between the PCR fragments derived from cDNA and gDNA templates was attributed to the presence of an intron within the gene fragment, which was later verified by DNA sequence comparison. From the lambda Uni-ZAP XR cDNA library prepared from 48-h A. parasiticus mRNA, approximately 150,000 plaques were screened. Sixteen positive cDNA clones were isolated and verified to be identical through restriction mapping. Approximately 150,000 plaques were screened from the lambda FixII gDNA library of A. parasiticus yielding six positive gDNA clones. Each was verified to contain vbs by PCR analysis, restriction mapping, Southern analysis, and/or nucleotide sequencing.Further investigations were undertaken with the genomic lambda clones, which successfully assigned the orientation of vbs and a probable cytochrome P450 monooxygenase (cyp) with respect to earlier portions of the putative aflatoxin gene cluster. Southern and PCR analysis positively identified the presence of omtA in two clones (λ56a and λ62b) and the absence of ver1 in all of the isolated gDNA clones. Although the vbs gene was verified by PCR to be present in each clone following library screening, subsequent examinations attested to a significant truncation of the 5′ terminus of vbs in two clones (λ52a and λ55c). Together, the genomic clones λ52a and λ62b contained approximately 30 kb of overlapping genomic sequence, as measured by restriction mapping (Fig. 5). The distance between vbs and omtA was measured by PCR and verified by DNA sequence analysis. Primer combinations Omt1-2NC + 56NC and Omt1-4NC + 56NC (Table II) gave 2.78- and 4.35-kb PCR products, respectively, which are in agreement with the known 1.49-kb separation between the Omt1 primers (66Yu J. Cary J.W. Bhatnagar D. Cleveland T.E. Keller N.P. Chu F.S. Appl. Environ. Microbiol. 1993; 59: 3564-3571Crossref PubMed Google Scholar). Employing the four omtA primers (Table I., Table II) of known orientations with primer 56NC (0.56 kb upstream of vbs), vbs and omtA were determined to be located within approximately 3.3 kb of each other, in opposite orientations (Fig. 5). Nucleotide sequence data from a 1.3-kb XbaI/KpnI genomic DNA fragment overlapping with the reported 5′ upstream region of omtA (J. Yu, 1993,) and an apparent cytochrome P450 monoxygenase,3 approximately 1400 bp in length, established the clustered nature of the three genes.The genomic nucleotide sequence of versicolorin B synthase has been determined and is contained within 2610 bp of phage clone λ62b. The transcribed cDNA clone possessed a continuous open reading frame of 1932 bp, as well as 20 bp of 5′-nontranslated and 161 bp of 3′-nontranslated regions. Comparison of the combined cDNA and genomic DNA sequences revealed that the coding region is interrupted by a single 53-bp intron (Fig. 6). The intervening sequence, which has been observed in other eukaryotic genes, shared the consensus regions 5′-(exon)/GTARGY … NRCTRAN … YAG/(exon)-3′ (68Boel E. Hansen M.T. Hjort I. Hpegh I. Fiil N.P. EMBO J. 1984; 3: 1581-1585Crossref PubMed Scopus (125) Google Scholar, 69Rambosek J.A. Leach J. Crit. Rev. Biotechnol. 1987; 6: 357-393Crossref PubMed Scopus (122) Google Scholar, 70Gurr S.J. Unkles S.E. Kinghorn J.R. Kinghorn J.R. Gene Structure in Eukaryotic Microbes. IRL Press, Oxford1990: 93Google Scholar). The Hogness box, TTTAAA, was seen −92 nucleotides from the vbs start codon. In addition, two putative CAAT promoter sequences (70Gurr S.J. Unkles S.E. Kinghorn J.R. Kinghorn J.R. Gene Structure in Eukaryotic Microbes. IRL Press, Oxford1990: 93Google Scholar) were detected at −162 and −224 nucleotides. A pyrimidine-rich motif, commonly associated with fungal promoters, was located between −72 and −60 nucleotides upstream of the start codon (70Gurr S.J. Unkles S.E. Kinghorn J.R. Kinghorn J.R. Gene Structure in Eukaryotic Microbes. IRL Press, Oxford1990: 93Google Scholar, 71Dobson M.J. Tuite M.F. Roberts N.A. Kingsman A.J. Kingsman S.M. Perkins R.E. Conroy S.C. Dunbar B. Fothegill L.A. Nucleic Acids Res. 1982; 10: 2625-2637Crossref PubMed Scopus (285) Google Scholar). A common trend found in this sequence and many other filamentous fungi genes was an adenine at the third nucleotide upstream of the start codon (70Gurr S.J. Unkles S.E. Kinghorn J.R. Kinghorn J.R. Gene Structure in Eukaryotic Microbes. IRL Press, Oxford1990: 93Google Scholar, 71Dobson M.J. Tuite M.F. Roberts N.A
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