Detailed Structure–Function Correlations of Bacillus subtilis Acetolactate Synthase |
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Authors: | Dr. Bettina Sommer Dr. Holger von Moeller Martina Haack Dr. Farah Qoura Clemens Langner Dr. Gleb Bourenkov Dr. Daniel Garbe Dr. Bernhard Loll Prof. Dr. Thomas Brück |
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Affiliation: | 1. Fachgebiet Industrielle Biokatalyse, Technische Universit?t München, Lichtenbergstrasse 4, 85748 Garching (Germany);2. Institut für Chemie und Biochemie, Abteilung Strukturbiochemie, Freie Universit?t Berlin, Takustrasse 6, 14195 Berlin (Germany);3. moloX GmbH, Takustrasse 6, 14195 Berlin (Germany);4. European Molecular Biology Laboratory, Notkestrasse 85, 22603 Hamburg (Germany) |
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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 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+ 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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Keywords: | biofuels biosynthesis biotechnology enzyme catalysis isobutanol |
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