Tricarballylic ester formation during biosynthesis of fumonisin mycotoxins in Fusarium verticillioides

Abstract

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 (DH₄-FB). MS and NMR analysis of DH₄-FB₁ indicated that the esters consist of aconitate with a 3'-alkene function, rather than a 2'-alkene function. Interestingly, the purified DH₄-FB₁ 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 DH₄-FB₁. The results suggest that DH₄-FB₁ 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 DH₄-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.

Keywords: Fusarium verticillioides; biosynthesis; fumonisins; mycotoxins.

Figure 1.

Chemical structure of fumonisins and analogs. DH₄–FB₁, tetradehydro fumonisin B1. (A) Structure of fumonism B1 and tetradehydro fumonism B1. (B) Example of possible regio- and sterioisomers of DH₄–FB₁.

Figure 2.

Isolation and analysis of DH₄–FB₁ from the FUM7 mutant. (A) HPLC analysis of crude extract; (B) HPLC analysis of the initially purified DH₄–FB₁; (C) HPLC analysis of the isomerized DH₄–FB₁; (D) MS analysis of the initially purified DH₄–FB₁; and (E) MS analysis of the isomerized DH₄–FB₁. A proposed cis–trans stereoisomerization of DH₄–FB₁ is included.

Figure 3.

SDS-PAGE of Fum7p produced in E. coli. Lane 1, purified Fum7p; Lane 2, size markers.

Figure 4.

MS analysis of the reaction mixtures containing Fum7p and tetradehydro fumonisins. (A) Standard fumonisins; (B) Fum7p + DH₄–FB + NADPH; (C) Fum7p + DH₄–FB + NADPH + FeCl₂; and (D) Fum7p + DH₄–FB + NADH + FeCl₂. The peaks corresponding to FB₁, FB_2/3, as well as the DH₄–FB analogs are indicted with arrows. The proposed structure for compounds 1, 2, and 3 are shown in Figure 5. 1, fumonisin analog with two oxalosuccinate esters; 2, fumonisin analog with one oxalosuccinate ester and one isocitrate ester; and 3, fumonisin analog with two isocitrate esters.

Figure 5.

A proposed mechanism for in vivo formation of tricarballylic esters in fumonisin biosynthesis and the observed in vitro activity of Fum7p. TCA, tricarboxylic acid; A, adenylation domain encoded by FUM10; PCP, peptidyl carrier protein domain in the protein encoded by FUM14; C, condensation domain in the protein encoded by FUM14; and HFB, hydrolyzed fumonisins.