Divergent Mechanistic Routes for the Formation of <i>gem</i>‐Dimethyl Groups in the Biosynthesis of Complex Polyketides

Abstract The gem ‐dimethyl groups in polyketide‐derived natural products add steric bulk and, accordingly, lend increased stability to medicinal compounds, however, our ability to rationally incorporate this functional group in modified natural products is limited. In order to characterize the mechanism of gem ‐dimethyl group formation, with a goal toward engineering of novel compounds containing this moiety, the gem ‐dimethyl group producing polyketide synthase (PKS) modules of yersiniabactin and epothilone were characterized using mass spectrometry. The work demonstrated, contrary to the canonical understanding of reaction order in PKSs, that methylation can precede condensation in gem ‐dimethyl group producing PKS modules. Experiments showed that both PKSs are able to use dimethylmalonyl acyl carrier protein (ACP) as an extender unit. Interestingly, for epothilone module 8, use of dimethylmalonyl‐ACP appeared to be the sole route to form a gem ‐dimethylated product, while the yersiniabactin PKS could methylate before or after ketosynthase condensation.