CLONING AND CHARACTERIZATION OF A CDNA FROM ASPERGILLUS-PARASITICUS ENCODING AN O-METHYLTRANSFERASE INVOLVED IN AFLATOXIN BIOSYNTHESIS

Aflatoxins are polyketide-derived secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. Among the catalytic steps in the aflatoxin biosynthetic pathway, the conversion of sterigmatocystin to O-methylsterigmatocystin and the conversion of dihydrosterigmatocystin to dihydro-O-methylsterigmatocystin are catalyzed by an S-adenosylmethionine-dependent O-methyltransferase. A cDNA library was constructed by using RNA isolated from a 24-h-old culture of wild-type A. parasiticus SRRC 143 and was screened by using polyclonal antiserum raised against a purified 40-kDa O-methyltransferase protein. A clone that harbored a full-length cDNA insert (1,460 bp) containing the 1,254-bp coding region of the gene omt-1 was identified by the antiserum and isolated. The complete cDNA sequence was determined, and the corresponding 418-amino-acid sequence of the native enzyme with a molecular weight of 46,000 was deduced. This 46-kDa native enzyme has a leader sequence of 41 amino acids, and the mature form of the enzyme apparently consists of 377 amino acids and has a molecular weight of 42,000. Direct sequencing of the purified mature enzyme from A. parasiticus SRRC 163 showed that 19 of 22 amino acid residues were identical to the amino acid residues in an internal region of the deduced amino acid sequence of the mature protein. The 1,460-bp omt-1 cDNA was cloned into an Escherichia coli expression system; a Western blot (immunoblot) analysis of crude extracts from this expression system revealed a 51-kDa fusion protein (fused with a 5-kDa beta-galactosidase N-terminal fragment). Furthermore, enzymatic activity assays of the E. coli crude extracts showed that sterigmatocystin was converted to 0-methylsterigmatocystin in the presence of S-adenosylmethionine. A 1.5-kb omt-1 gene transcript was detected by Northern (RNA) blot analysis in total RNAs isolated from submerged A. parasiticus cultures grown in a medium which induces aflatoxin B, production that were 24, 48, 72, and 96 h old but not in RNA from a culture that was 18 h old. Transcript accumulation correlated well with the increased rate of aflatoxin accumulation in these cultures. A comparison of the predicted amino acid sequence of O-methyltransferase with previously described sequences revealed a proposed S-adenosylmethionine-binding site motif found in other S-adenosylmethionine-dependent methyltransferases.