Enzymological evidence for separate pathways for aflatoxin B1 and B2 biosynthesis

Abstract

Aflatoxins B1 (AFB1) and B2 (AFB2) are biologically active secondary metabolites of Aspergillus flavus and Aspergillus parasiticus. These toxins are synthesized by the fungi from pathway precursors: sterigmatocystin (ST)----O-methylsterigmatocystin (OMST)----AFB1; dihydrosterigmatocystin (DHST)----dihydro-O-methylsterigmatocystin (DHOMST)----AFB2. The late stages of AFB1 synthesis are carried out by two enzyme activities, a methyltransferase (MT) (ST----OMST), and an oxidoreductase (OR) (OMST----AFB1). Properties of the purified MT have been identified in a previous investigation [Bhatnagar et al. (1988) Prep. Biochem. 18, 321]. In the current study, the OR was partially purified (150-fold of specific activity) from fungal cell-free extracts and characterized with extended investigation of the late stages of AFB1 and AFB2 synthesis. Whole cells of an isolate of A. flavus (SRRC 141), which produce only AFB2, were able to produce AFB1 in ST and OMST feeding studies; the results suggested that the enzymes involved in AFB2 biosynthesis also carry out AFB1 synthesis. Substrate competition experiments carried out with the OR showed that an increasing concentration of either OMST or DHOMST in the presence of a fixed, nonsaturating concentration of either DHOMST or OMST, respectively, resulted in a decline in production of one aflatoxin (B1 or B2) with a corresponding increase in the synthesis of the other toxin (B2 or B1). OMST was a preferred substrate (Km, 1.2 microM) for the oxidoreductase as compared to DHOMST (Km, 13.4 microM). Similar, substrate competition experiments showed that ST (Km, 2.0 microM) was a preferred substrate over DHST (Km, 22.5 microM) for a homogeneous MT.(ABSTRACT TRUNCATED AT 250 WORDS)