Enzymatic formation of G-group aflatoxins and biosynthetic relationship between G- and B-group aflatoxins

We detected biosynthetic activity for aflatoxins G(1) and G(2) in cell extr acts of Aspergillus parasiticus NIAH-26. We found that in the presence of N ADPH, aflatoxins G(1) and G(2) were produced from O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin, respectively. No G-group aflatoxins w ere produced from aflatoxin B-1, aflatoxin B-2, 5-methoxysterigmatocystin, dimethoxysterigmatocystin, or sterigmatin, confirming that B-group aflatoxi ns are not the precursors of G-group aflatoxins and that G- and B-group afl atoxins are independently produced from the same substrates (O-methylsterig matocystin and dihydro-O-methylsterigmatocystin). In competition experiment s in which the cell-free system was used, formation of aflatoxin G(2) from dihydro-O-methylsterigmatocystin was suppressed when O-methylsterigmatocyst in was added to the reaction mixture, whereas aflatoxin G(1) was newly form ed. This result indicates that the same enzymes can catalyze the formation of aflatoxins G(1) and G(2). Inhibition of G-group aflatoxin formation by m ethyrapone, SKF-525A, or imidazole indicated that a cytochrome P-450 monoox ygenase may be involved in the formation of G-group aflatoxins. Both the mi crosome fraction and a cytosol protein with a native mass of 220 kDa were n ecessary for the formation of G-group aflatoxins, Due to instability of the microsome fraction, G-group aflatoxin formation was less stable than B-gro up aflatoxin formation. The ordA gene product, which may catalyze the forma tion of B-group aflatoxins, also may be required for G-group aflatoxin bios ynthesis, We concluded that at least three reactions, catalyzed by the ordA gene product, an unstable microsome enzyme, and a 220-kDa cytosol protein, are involved in the enzymatic formation of G group aflatoxins from either O-methylsterigmatocystin or dihydro-O-methylsterigmatocystin.