Detailed Structure–Function Correlations of Bacillus Subtilis Acetolactate Synthase
ChemBioChem(2015)
Abstract
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 Mg 2+ 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, Mg 2+ 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 residues.
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Key words
biofuels,biosynthesis,biotechnology,enzyme catalysis,isobutanol
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