Tricarballylic ester formation during biosynthesis of fumonisin mycotoxins in Fusarium verticillioides

Fumonisins are agriculturally important mycotoxins produced by the maize pathogen Fusarium verticillioides. The chemical structure of fumonisins contains two tricarballylic esters, which are rare structural moieties and important for toxicity. The mechanism for the tricarballylic ester formation is not well understood. FUM7 gene of F. verticillioides was predicted to encode a dehydrogenase/reductase, and when it was deleted, the mutant produced tetradehydro fumonisins (DH(4)–FB). MS and NMR analysis of DH(4)–FB(1) indicated that the esters consist of aconitate with a 3′-alkene function, rather than a 2′-alkene function. Interestingly, the purified DH(4)–FB(1) eventually yielded three chromatographic peaks in HPLC. However, MS revealed that the metabolites of the three peaks all had the same mass as the initial single-peak DH(4)–FB(1). The results suggest that DH(4)–FB(1) can undergo spontaneous isomerization, probably including both cis–trans stereoisomerization and 3′- to 2′-ene regioisomerization. In addition, when FUM7 was expressed in Escherichia coli and the resulting enzyme, Fum7p, was incubated with DH(4)–FB, no fumonisin with typical tricarballylic esters was formed. Instead, new fumonisin analogs that probably contained isocitrate and/or oxalosuccinate esters were formed, which reveals new insight into fumonisin biosynthesis. Together, the data provided both genetic and biochemical evidence for the mechanism of tricarballylic ester formation in fumonisin biosynthesis.