Abstract
Aflatoxins, the most toxic and carcinogenic family of fungal secondary metabolites, are frequent contaminants of foods intended for human consumption. Previous studies showed that formation of G-group aflatoxins (AFs) from O-methylsterigmatocystin (OMST) by certain Aspergillus species involves oxidation by the cytochrome P450 monooxygenases, OrdA (AflQ) and CypA (AflU). However, some of the steps in the conversion have not yet been fully defined. Extracts of Aspergillus parasiticus disruption mutants of the OYE-FMN binding domain reductase-encoding gene nadA (aflY) contained a 386 Da AFG1 precursor. A compound with this mass was predicted as the product of sequential OrdA and CypA oxidation of OMST. Increased amounts of a 362 Da alcohol, the presumptive product of NadA reduction, accumulate in extracts of fungi with disrupted aryl alcohol dehydrogenase-encoding gene norB. These results show that biosynthesis of AFG1 involves NadA reduction and NorB oxidation.
Highlights
Aflatoxins (AFs) are Aspergillus secondary metabolites that are highly toxic to certain animal species, in particular, birds and fish, and are potent carcinogens [4]
Disruption of nadA, norA or norB was achieved by transformation of A. parasiticus BN9Δku70 with the insert portion of plasmid constructs in which the A. oryzae pyrithiamine reductase gene, ptr, displaced part of the coding region of each gene
When the greenish-blue fluorescent compound (GFC) was completely separated from AFG1 by preparative Thin layer chromatography (TLC), a small amount of AFG1 still appeared in the product mixture (Figure 2B, lane 2)
Summary
Aflatoxins (AFs) are Aspergillus secondary metabolites that are highly toxic to certain animal species, in particular, birds and fish, and are potent carcinogens [4]. 11-HydroxyOMST (HOMST) was proven to be a precursor of AFB1 [13] and is the expected product from OrdA-catalyzed oxidization of the A-ring of OMST (Figure 1). Because expression of ordA in yeast enabled the yeast to convert OMST to AFB1, it was assumed that OrdA catalyzes the oxidation of HOMST This oxidation is expected to produce a 370 Da intermediate 1 or 2 Showed that nadA encodes an enzyme that is involved in formation of AFG1 and isolated an intermediate with mass 360 Da from nadA mutant cultures. They describe this compound as the immediate AFG1 precursor [3]. NorB is responsible for the final oxidation step in AFG1 formation
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