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Title: Biocatalysis of fumarate derivatives by flavocytochrome c3
Author: Wardrope, Caroline
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2007
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Flavocytochrome c3 (Fcc3) is a 63.8 kDa soluble fumarate reductase produced by the bacterium Shewanella frigidimarina.  The aim of this project was to investigate the ability of Fcc3 to reduce alternative substrates, particularly those that may result in the production of chiral molecules. Fcc3 is able to reduce 2-methylfumarate to 2-methylsuccinate in vitro, with a kcat of 8.97.s-1 ± 0.43, and a Km of 31.7 μM ± 8.5 at pH 7.2. Circular dichroism spectroscopy revealed that reduction of 2-methylfumarate by wild-type Fcc3 is stereospecific, producing S-methylsuccinate at >95% enantiomeric excess. The crystal structure of Wild-type Fcc3 with 2-methylfumarate bound was solved to 1.5 Å and showed that the mode of 2-methylfumarate binding to the active site always results in the production of S-methylsuccinate. A range of fumarate derivatives were tested as potential substrates but wild-type Fcc3 did not catalyse the reduction of anything other than fumarate and 2-methylfumarate. In order to find out if the substrate specificity of Fcc3 could be altered, active site residues involved in Hydrogen-bonding with substrate were substituted by site-directed mutagenesis. Subsequent kinetic studies demonstrated that most of the mutants constructed were still able to reduce fumarate and 2-methylfumarate, although at rates differing from that measured for wild-type Fcc3. Crystal structures were obtained for the mutants T377A and H365G at 2.0 Å and 1.9 Å respectively. Previous theoretical modelling studies had predicted that some mutants (especially H365G) may be able to catalyse the reduction of some mono-acids. However, it appears that this is not the case as none of the engineered forms of Fcc3 were able to reduce any substrates other than fumarate and 2-methylfumarate. Fcc3 is therefore a highly substrate-specific fumarate reductase.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available