ISOLATION AND CHARACTERIZATION OF THE VERSICOLORIN-B SYNTHASE GENE FROM ASPERGILLUS-PARASITICUS - EXPANSION OF THE AFLATOXIN B-1 BIOSYNTHETIC GENE-CLUSTER

Versicolorin B synthase catalyzes the side chain cyclizatian of racemi c versiconal hemiacetal (7) to the bisfuran ring: system of (-)-versic olorin B (8), an essential transformation in the aflatoxin biosyntheti c pathway of Aspergillus parasiticus. The dihydrobisfuran an is key to the mutagenic nature of aflatoxin B-1 (1), The protein, which skews 5 8% similarity and 38% identity with glucose oxidase from Aspergillus n iger, possesses an amino-terminal sequence homologous to the ADP-bindi ng region of other flavoenzymes. However, this enzyme does not require flavin or nicotinamide cofactors for its cyclase activity, The 643-am ino acid native enzyme contains three potential sites for W-linked gly cosylation, Asn-Xaa-Thr or Asn-Xaa-Ser. The cDNA and genomic clones of versicolorin B synthase were isolated by screening the respective lib raries with random-primed DNA probes generated from an exact copy of a n internal vbs sequence. This probe was created through polymerase cha in reaction by using nondegenerate polymerase chain reaction primers d erived from the amino acid sequences of peptide fragments of the enzym e. The 1985-base genomic rbs DNA sequence is interrupted by one intron of 53 nucleotides. Southern blotting, nucleotide sequencing, and deta iled 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 obs supports the theory that the genes encoding the aflatoxin biosynthetic enzymes in A , parasiticus are clustered.