Geometric isomerization can expand the scope of biological activities of natural products. The observed chemical diversity among the pseurotin-type fungal secondary metabolites is in part generated by a trans to cis isomerization of an olefin. In vitro characterizations of pseurotin biosynthetic enzymes revealed that the glutathione S-transferase PsoE requires participation of the bifunctional C-methyltransferase/epoxidase PsoF to complete the trans to cis isomerization of the pathway intermediate presynerazol. The crystal structure of the PsoE/glutathione/presynerazol complex indicated stereospecific glutathione-presynerazol conjugate formation is the principal function of PsoE. Moreover, PsoF was identified to have an additional, unexpected oxidative isomerase activity, thus making it a trifunctional enzyme which is key to the complexity generation in pseurotin biosynthesis. Through the study, we identified a novel mechanism of accomplishing a seemingly simple trans to cis isomerization reaction.