Marlow, Victoria A. (2013) Mechanistic studies of acetolactate decarboxylase. PhD thesis, University of Warwick.

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Abstract

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.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Microbial enzymes, Decarboxylases, Decarboxylation
Official Date:	June 2013
Institution:	University of Warwick
Theses Department:	Molecular Organisation and Assembly in Cells
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Wills, Martin; Fülöp, Vilmos
Sponsors:	Engineering and Physical Sciences Research Council (EPSRC)
Extent:	xxii, 221 leaves : illustrations.
Language:	eng

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