Submitted to: Journal of Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/17/2005
Publication Date: 2/28/2006
Citation: Zaleta-Rivera, K., Xu,C., Yu, F., Butchko, R.A.E., Proctor, R.H., Lara, M.E.H., Raza, A., Dussault, P.H., Du, L. 2006. A bidomain nonribosomal peptide synthetase encoded by FUM14 catalyzes the formation of tricarballylic esters in the biosynthesis of fumonisin. Journal of Biochemistry. 45:2561-2569. Interpretive Summary: Fumonisins are toxic substances produced by the fungus Gibberella moniliformis. The fungus can cause ear and stalk rot of corn and can contaminate the corn with fumonisins. We are studying the biology of the fungus, including the genetics and biochemistry associated with fumonisin production, in an effort to reduce or eliminate these toxins from corn. It has been suggested that there are a number of steps involved in the production of fumonisins. We have identified a number of genes involved in the production of fumonisins. In this report, we characterize in detail the nature of one of these genes and its role in fumonisin production. This was accomplished in two ways. First, the function of the gene was eliminated by removing the gene from the fungus. We then studied the effect of removing this gene on the production of fumonisin. As we predicted, the fungus could no longer produce fumonisin correctly, but produced a derivative form of fumonisin. This experiment provided two important pieces of information. One, that the gene is involved in fumonisin production and two, that the chemical structure of the fumonisin derivative indicates what role the gene plays in fumonisin production. The second way this gene was characterized involved isolating the product of the gene. The product of the gene is a protein which acts as an enzyme which performs a specific chemical reaction. By combining the purified protein with the predicted chemicals that it needs to perform the specific chemical reaction and then characterizing the resulting chemical reaction, we learned that the gene produces a protein which performs the chemical reaction needed to produce fumonisin. We can use this understanding of the chemical pathway of fumonisin production to devise novel ways to try to eliminate or reduce fumonisin contamination of corn.
Technical Abstract: Fumonisins are a group of polyketide-derived mycotoxins produced by Fusarium verticillioides, a filamentous fungus infecting corn and contaminating food and feeds. The metabolites contain two tricarballylic esters, which are critical to the toxicity of the mycotoxins. Here, we present genetic and biochemical data for the esterification mechanism. FUM14 in F. verticillioides has been deleted by homologous recombination, and the resultant mutant lost the ability to produce fumonisins. Two metabolites, HFB3 and HFB4, which are biosynthetic precursors of fumonisins lacking the tricarballylic esters, were detected in the mutant. The results suggest that FUM14 is required for the esterification of fumonisins. FUM14 was predicted to encode a nonribosomal peptide synthetase (NRPS) containing two domains, peptidyl carrier protein and condensation domain. Both the intact Fum14p and the condensation domain have been expressed in E. coli and purified for activity assays. The data showed that Fum14p was able to convert HFB3 and HFB4 to FB3 and FB4, respectively, when incubated with tricarballylic thioester of N-acetylcysteamine. In addition, the condensation domain was able to convert HFB1 to FB1. The biochemical data provide direct evidence for the role of FUM14 in the esterification of fumonisins. More interestingly, the results reveal the first example for an NRPS condensation domain to catalyze a C-O bond (ester) formation, instead of the typical C-N bond (amide) formation in nonribosomal peptides. The understanding of the esterification mechanism provides useful knowledge for mycotoxin reduction and elimination. The study also provides new insight into the reactions catalyzed by NRPS.