Detailed structure-function correlations of bacillus subtilis acetolactate synthase

Isobutanol is deemed to be a next-generation biofuel and a renewable platform chemical.^[1] Non-natural biosynthetic pathways for isobutanol production have been implemented in cell-based and in vitro systems with Bacillus subtilis acetolactate synthase (AlsS) as key biocatalyst.^[2-6] AlsS catalyzes the condensation of two pyruvate molecules to acetolactate with thiamine diphosphate and Mg²⁺ as cofactors. AlsS also catalyzes the conversion of 2-ketoisovalerate into isobutyraldehyde, the immediate precursor of isobutanol. Our phylogenetic analysis suggests that the ALS enzyme family forms a distinct subgroup of ThDP-dependent enzymes. To unravel catalytically relevant structure-function relationships, we solved the AlsS crystal structure at 2.3 Å in the presence of ThDP, Mg²⁺ in a transition state with a 2-lactyl moiety bound to ThDP. We supplemented our structural data by point mutations in the active site to identify catalytically important residues.