The in Vitro Conversion of Norsolorinic Acid to Aflatoxin B1. An Improved Method of Cell-Free Enzyme Preparation and Stabilization

Abstract

The biosynthesis of the environmental carcinogen aflatoxin B1 (13) is initiated by the formation of a C6-primer by a dedicated yeast-like fatty acid synthase. Homologation of this starter unit by a polyketide synthase gives the anthraquinone norsolorinic acid (2). Approximately 15 chemical steps follow from this first stable intermediate to the mycotoxin (13) itself. A new protocol of cell-free enzyme preparation has been developed from the fungus Aspergillus parasiticus which carries out all of these transformations for the first time. The key experimental step involves rapid concentration and efficient dialysis by membrane filtration to remove primary and secondary metabolites, cofactors, and small biomolecules (MW < 10 000). All enzymes of the aflatoxin biosynthetic pathway have been dramatically stabilized by this procedure, and the effects of added substrates and cofactors can be assayed against virtually no background reactions. The overall pathway from norsolorinic acid (2) to aflatoxin B1 (13) has been investigated, cofactor requirements defined for each step, and a time-course run in which only versicolorin A (9) and sterigmatocystin (11) were observed to accumulate. The post-bisfuran skeletal rearrangement of versicolorin A (9) to demethylsterigmatocystin (10) was studied in O-methylsterigmatocystin (12) in the presence of D2O and/or d7-glucose or stereospecifically labeled NADPD. Unexpectedly high extents of proton exchange were found in the A ring during this transformation, including at a site of formal reduction. A tentative mechanism is discussed to account for this multi enzyme process.