Biochemical and structural characterisation of a thermophilic Aldo-Keto Reductase fromThermotoga maritima
Date: 1 March 2009
Thesis or dissertation
University of Exeter
PhD in Biosciences
The Aldo-Keto Reductases (AKR) are a group of oxidoreductase enzymes structurally and mechanistically distinct from the Alcohol Dehydrogenases (ADH). The AKRs are of importance for their ability to produce industrially useful compounds including chiral secondary alcohols. The ADH family have traditionally been exploited for chiral ...
The Aldo-Keto Reductases (AKR) are a group of oxidoreductase enzymes structurally and mechanistically distinct from the Alcohol Dehydrogenases (ADH). The AKRs are of importance for their ability to produce industrially useful compounds including chiral secondary alcohols. The ADH family have traditionally been exploited for chiral alcohol production; the AKR family have currently been underexploited for chiral alcohol production and present the opportunity to search for novel oxidoreductases with properties and substrate specificities distinct from the ADH enzymes. The AKR studied here, from the hyperthermophilic bacteria Thermotoga maritima has been characterised with respect to its biochemical and structural properties, and its potential as a biocatalyst evaluated. This enzyme is the second example of a thermophilic AKR to have its three dimensional structure solved, the other also being from Thermot. maritima. The AKR studied exhibits high stability with respect to temperature and moderate amounts of organic solvents. A large preference for the reduction reaction compared to the oxidation reaction was found, which has previously been observed in other AKRs. The X-ray crystal structure was solved to 2.6Å resolution in the apo form. The final structure has three loop sections which were not located due to disorder within the crystal, which are expected to become ordered upon cofactor and substrate binding. A section of one of these missing loops was found to bind at the active site of the enzyme, with a glutamate occupying the site of substrate carbonyl binding. The formation of a dimer, increased helix-dipole stabilisation and long distance ion pair interactions all act to increase thermostability of the AKR with respect to its mesophilic homologues. The X-ray crystal structure of Escherichia coli bacterioferritin has also been solved to 1.9Å resolution, which was co-purified along with the recombinant AKR enzyme. This structure shows the symmetrical binding of a heme molecule on the local two-fold axis between subunits and the binding of two metal atoms to each subunit at the ferroxidase centre. These metal atoms have been identified as zinc by the anaylsis of the structure and X-ray data and confirmed by microPIXE experiments. For the first time the heme has been shown to be linked to the internal and external environments via a cluster of waters positioned above the heme molecule. This information has provided a greater insight into the function and mechanism of bacterioferritin.
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