Characterization of the critical amino acids of an Aspergillus parasiticuscytochrome P-450 monooxygenase encoded by ordA that is involved in the biosynthesis of aflatoxins B-1, G(1), B-2, and G(2)

The conversion of O-methylsterigmatocystin (OMST) and dihydro-O-methylsteri gmatocystin to a aflatoxins B-1, G(1), B-2, and G(2) requires a cytochrome P-450 type of oxidoreductase activity. ordA, a gene adjacent to the omtA ge ne, was identified in the aflatoxin-biosynthetic pathway gene cluster by ch romosomal walking in Aspergillus parasiticus, The ordA gene was a homolog o f the Aspergillus flavus ord1 gene, which is involved in the conversion of OMST to aflatoxin B-1. Complementation of A. parasiticus SRRC 2043, an OMST -accumulating strain, with the ordA gene restored the ability to produce af latoxins B-1, G(1), B-2, and G(2). The ordA gene placed under the control o f the GAL1 promoter converted exogenously supplied OMST to aflatoxin B-1 in Saccharomyces cerevisiae. In contrast, the ordA gene homolog in A. parasit icus SRRC 2043, ordA1, was not able to carry out the same conversion in the yeast system. Sequence analysis revealed that the ordA1 gene had three poi nt mutations which resulted in three amino acid changes (His-400-->Leu-400, Ala-143-->Ser-143, and Ile-528-->Tyr-528). Site-directed mutagenesis studi es showed that the change of His-400 to Leu-400 resulted in a loss of the m onooxygenase activity and that Ala-143 played a significant role in the cat alytic conversion, In contrast, Ile-528 was not associated with the enzymat ic activity, The involvement of the ordA gene in the synthesis of aflatoxin s G(1), and G(2) in A. parasiticus suggests that enzymes required fur the f ormation of aflatoxins G(1) and G(2) are not present in A. flavus. The resu lts showed that in addition to the conserved heme-binding and redox reactio n domains encoded by ordA, other seemingly domain-unrelated amino acid resi dues are critical for cytochrome P-450 catalytic activity. The ordA gene ha s been assigned to a new cytochrome P-450 gene family named CYP64 by The Cy tochrome P450 Nomenclature Committee.