HOMST was implicated as a potential intermediate in synthetic feeding studies with either A. parasiticus cultures or with yeast expressing ordA (Udwary et al., 2002), and this intermediate was confirmed here in our product analysis. Our results indicate that NorA is involved in a catalytic step after OrdA oxidation and are consistent with the route proposed

in Fig. 5, where OrdA is predicted to catalyze oxidation of HOMST to a putative 370 Da lactone. The subsequent rearrangement steps of the presumptive 370 Da lactone Selleckchem BI-2536 are less clear. Ultimately, these are likely to result in the formation of the 326 Da methyl enolether shown in Fig. 5, which is likely to be the immediate AFB1 precursor. Recent results suggest that the aflatoxin biosynthesis gene, hypE, encodes a protein with an EthD domain that may be involved in the oxidative demethylation of this methyl enolether (Holmes, 2008). Proteins with an EthD domain, previously only reported in bacteria, are required for oxidative ethyl-tert-butyl ether

degradation in the presence of a cytochrome P450 monooxygenase (Chauvaux et al., 2001). Disruption of hypE in A. flavus led to accumulation of a compound with the intense blue fluorescence characteristic of deoxyAFB1 and aflatoxins, but that migrated faster than AFB1 on TLC. This new metabolite exhibited a mass of 328 Da, which is consistent with the methyl ether shown in Fig. 5. Oxidation of the methyl ether in either the 326 or the 328 Da intermediates may occur with HypE and an unknown NVP-LDE225 order cytochrome P450 enzyme [possibly OrdA or CypX Sitaxentan (AflV)] to cause loss of the methyl as formaldehyde and directly yield AFB1 or AFOH, respectively. AFOH resulting from demethylation of the 328 Da ether would require NorA-catalyzed oxidation to AFB1. In the absence of NorA, the 326 Da methyl enolether

or AFB1 may be partially reduced to the 328 Da methyl ether in the reductive metabolic environment of the cell as shown in Fig. 5. As suggested previously, the formation of increased quantities of deoxyAFB1 rather than AFOH in the absence of NorA could be a consequence of the precursor metabolites being produced and isolated under acidic culture conditions. In our studies, synthetic AFOH was found to dehydrate readily under mild acidic conditions. In the fungal cell, the pH is likely to be significantly higher and therefore, if AFOH is formed, it is unlikely that it would be subjected to acid-catalyzed dehydration. The balance in the cellular environment between oxidation and reduction as well as the availability of active transport out of the cell of AFB1 would be expected to play critical roles in determining the levels of the individual precursors and in maintaining the oxidation state of AFB1.