Mechanistic Insights into the Substrate Promiscuity of α-Ketoacid Decarboxylases for Enhanced Higher-Alcohol Biosynthesis

α-Ketoacid decarboxylases (KDCs) are key enzymes in higher alcohol biosynthesis, but their inherent substrate promiscuity limits product selectivity and synthetic efficiency. Here, we report crystal structures of a thiamine diphosphate (ThDP)-dependent KDC in complex with multiple substrates. Structural analysis reveals an oxyanion hole─formed by a conserved HH-motif and the 4′-amino group of ThDP─that anchors the α-ketoacid moiety to facilitate catalysis. A spacious, hydrophobic substrate-binding pocket accommodates diverse substrate tails, underlying the enzyme’s broad specificity. Theoretical calculations further clarified the kinetic differences in substrate-dependent catalysis. Guided by these insights, we rationally redesigned the substrate-binding pocket to enhance discrimination between phenylpyruvate and indole-3-pyruvate. The engineered KDC variant Q383E enhanced 2-phenylethanol (2-PE) production in Escherichia coli, achieving a 16% increase in conversion efficiency and a titer of 27.9 g/L in fermentation. This work provides a mechanistic framework for tailoring KDC specificity and optimizing microbial pathways for higher alcohol biosynthesis.