Understanding the order of methylation and condensation in geminal-dimethyl producing polyketide synthase domains

Methyl transfer in geminal-dimethyl producing type I polyketide synthases (PKS) has not been rigorously studied.  The methyl transfer reaction in type I polyketide synthases is presumed to occur after the extension reaction performed by the ketosynthase, but this reaction order has not been experimentally demonstrated. We sought to understand the order of extension and methylation in the yersiniabactin PKS and in module 8 of the epothilone cluster to develop a mechanistic understanding of PKS catalyzed methylation. Tandem and high-resolution mass spectrometry techniques allowed us to examine acyl intermediates formed during the course of in vitro reactions with gem-dimethyl producing PKSs.