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Title: Mechanistic studies of acetolactate decarboxylase
Author: Marlow, Victoria A.
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2013
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Acetolactate decarboxylase (ALDC) is a bacterial enzyme of the butanediol fermentation pathway that decarboxylates (S)-acetolactate into (R)-acetoin. Remarkably this enzyme also catalyses the decarboxylation of the opposite enantiomer, (R)-acetolactate, to give the same product, (R)-acetoin. It is unusual for an enzyme to convert racemic substrate into an enantiomerically pure product. This unusual stereochemical control has led to extensive study of the ALDC mechanism and the hypothesis that ALDC catalyses the rearrangement of (R)-acetolactate into (S)-acetolactate prior to decarboxylation. The research presented in this thesis sought to gain insight into the molecular mechanism of the ALDC catalysed reaction by using a combination of kinetic and structural techniques. Bacillus subtilis alsD encoding ALDC was cloned into an expression vector and a series of active site mutants were prepared. The activity of mutant AlsD were determined using a circular dichroism based assay, which identified that the two active site glutamates and a basic residue are required for catalysis. A series of chiral transition state analogues were prepared in a two-step synthesis to give enantiomerically enriched 2,3-dihydroxy-2-methylbutanoic acid in reasonable yields. Three of the compounds were identified as competitive inhibitors, co-crystallised with Bacillus brevis ALDC and structures solved to 1.1-1.6 Å. These structures, coupled with inhibition studies and site-directed mutagenesis, provide an improved understanding of the molecular processes involved in the stereoselective decarboxylation of acetolactate. A mechanism for the transformation of each enantiomer of acetolactate is proposed.
Supervisor: Not available Sponsor: Engineering and Physical Sciences Research Council (EPSRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QR Microbiology