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.
Keywords:
biofuels; biosynthesis; biotechnology; enzyme catalysis; isobutanol.
© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
MeSH terms
-
Acetolactate Synthase / chemistry*
-
Acetolactate Synthase / genetics
-
Acetolactate Synthase / metabolism
-
Aldehydes / chemistry
-
Aldehydes / metabolism
-
Bacillus subtilis / chemistry*
-
Bacillus subtilis / classification
-
Bacillus subtilis / enzymology
-
Bacillus subtilis / genetics
-
Bacterial Proteins / chemistry*
-
Bacterial Proteins / genetics
-
Bacterial Proteins / metabolism
-
Biocatalysis
-
Biofuels
-
Butanols / chemistry*
-
Butanols / metabolism
-
Catalytic Domain
-
Cations, Divalent
-
Coenzymes / chemistry
-
Coenzymes / metabolism
-
Crystallography, X-Ray
-
Escherichia coli / genetics
-
Escherichia coli / metabolism
-
Gene Expression
-
Hemiterpenes
-
Keto Acids / chemistry
-
Keto Acids / metabolism
-
Lactates / chemistry
-
Lactates / metabolism
-
Magnesium / chemistry
-
Magnesium / metabolism
-
Models, Molecular
-
Phylogeny
-
Point Mutation
-
Pyruvic Acid / chemistry
-
Pyruvic Acid / metabolism
-
Structure-Activity Relationship
-
Thiamine Pyrophosphate / chemistry
-
Thiamine Pyrophosphate / metabolism
Substances
-
Aldehydes
-
Bacterial Proteins
-
Biofuels
-
Butanols
-
Cations, Divalent
-
Coenzymes
-
Hemiterpenes
-
Keto Acids
-
Lactates
-
alpha-acetolactate
-
isobutyl alcohol
-
alpha-ketoisovalerate
-
Pyruvic Acid
-
isobutyraldehyde
-
Acetolactate Synthase
-
Magnesium
-
Thiamine Pyrophosphate