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 in the presence of ThDP, Mg2 and 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 residue