Enzymatic Formation of G-Group Aflatoxins and Biosynthetic Relationship between G- and B-Group Aflatoxins

Abstract

ABSTRACT We detected biosynthetic activity for aflatoxins G 1 and G 2 in cell extracts of Aspergillus parasiticus NIAH-26. We found that in the presence of NADPH, aflatoxins G 1 and G 2 were produced from O -methylsterigmatocystin and dihydro- O -methylsterigmatocystin, respectively. No G-group aflatoxins were produced from aflatoxin B 1 , aflatoxin B 2 , 5-methoxysterigmatocystin, dimethoxysterigmatocystin, or sterigmatin, confirming that B-group aflatoxins are not the precursors of G-group aflatoxins and that G- and B-group aflatoxins are independently produced from the same substrates ( O -methylsterigmatocystin and dihydro- O -methylsterigmatocystin). In competition experiments in which the cell-free system was used, formation of aflatoxin G 2 from dihydro- O -methylsterigmatocystin was suppressed when O -methylsterigmatocystin was added to the reaction mixture, whereas aflatoxin G 1 was newly formed. 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 methyrapone, SKF-525A, or imidazole indicated that a cytochrome P-450 monooxygenase may be involved in the formation of G-group aflatoxins. Both the microsome fraction and a cytosol protein with a native mass of 220 kDa were necessary for the formation of G-group aflatoxins. Due to instability of the microsome fraction, G-group aflatoxin formation was less stable than B-group aflatoxin formation. The ordA gene product, which may catalyze the formation of B-group aflatoxins, also may be required for G-group aflatoxin biosynthesis. 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.