Zhang_2021_Chemistry_10_3106

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. Here 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 .