Chemical and Genetic Studies on the Formation of Pyrrolones During the Biosynthesis of Cytochalasans

A key step during the biosynthesis of cytochalasans is a proposed Knoevenagel condensation to form the pyrrolone core, enabling the subsequent 4+2 cycloaddition reaction that results in the characteristic octahydroisoindolone motif of all cytochalasans. In this work, we investigate the role of the highly conserved alpha,beta-hydrolase enzymes PyiE and ORFZ during the biosynthesis of pyrichalasin H and the ACE1 metabolite, respectively, using gene knockout and complementation techniques. Using synthetic aldehyde models we demonstrate that the Knoevenagel condensation proceeds spontaneously but results in the 1,3-dihydro-2H-pyrrol-2-one tautomer, rather than the required 1,5-dihydro-2H-pyrrol-2-one tautomer. Taken together our results suggest that the alpha,beta-hydrolase enzymes are essential for first ring cyclisation, but the precise nature of the intermediates remains to be determined.

Automated summary

This study investigates the role of alpha,beta-hydrolase enzymes during the biosynthesis of cytochalasans, specifically focusing on their involvement in the first ring cyclisation. It was found that the Knoevenagel condensation proceeds spontaneously but leads to the formation of a different tautomer than the one required for the subsequent reactions. Further research is needed to determine the precise nature of the intermediates.