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 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.
Fachgebiet Industrielle Biokatalyse, Technische Universität München, Lichtenbergstrasse 4, 85748 Garching (Germany).
Bettina
B
Sommer
Holger
H
von Moeller
Martina
M
Haack
Farah
F
Qoura
Clemens
C
Langner
Gleb
G
Bourenkov
Daniel
D
Garbe
Bernhard
B
Loll
Thomas
T
Brück
D000095:Acetolactate Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000447:Aldehydes / Q000737:chemistry / Q000378:metabolism; D001412:Bacillus subtilis / Q000737:chemistry* / Q000145:classification / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D056804:Biofuels; D000440:Butanols / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D002413:Cations, Divalent; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D045782:Hemiterpenes; D007651:Keto Acids / Q000737:chemistry / Q000378:metabolism; D007773:Lactates / Q000737:chemistry / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010802:Phylogeny; D017354:Point Mutation; D019289:Pyruvic Acid / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013835:Thiamine Pyrophosphate / Q000737:chemistry / Q000378:metabolism
D000447:Aldehydes; D001426:Bacterial Proteins; D056804:Biofuels; D000440:Butanols; D002413:Cations, Divalent; D003067:Coenzymes; D045782:Hemiterpenes; D007651:Keto Acids; D007773:Lactates; C006359:alpha-acetolactate; C040507:isobutyl alcohol; C001505:alpha-ketoisovalerate; D019289:Pyruvic Acid; C017439:isobutyraldehyde; D000095:Acetolactate Synthase; D008274:Magnesium; D013835:Thiamine Pyrophosphate
biofuels; biosynthesis; biotechnology; enzyme catalysis; isobutanol