Biochemical and structural characterisation of a thermophilic Aldo-Keto Reductase fromThermotoga maritima

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Biochemical and structural characterisation of a thermophilic Aldo-Keto Reductase fromThermotoga maritima

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dc.contributor.author Simon, Willies en_GB
dc.date.accessioned 2009-03-24T14:27:45Z en_GB
dc.date.accessioned 2011-01-26T08:33:25Z en_US
dc.date.accessioned 2013-03-21T10:09:53Z
dc.date.issued 2009-03 en_GB
dc.description.abstract 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. en_GB
dc.description.sponsorship BBSRC en_GB
dc.description.sponsorship Chirotech Technology Ltd. en_GB
dc.identifier.uri http://hdl.handle.net/10036/57013 en_GB
dc.language.iso en en_GB
dc.publisher University of Exeter en_GB
dc.subject aldo-keto reductase en_GB
dc.subject X-ray crystallography en_GB
dc.subject Bacterioferritin en_GB
dc.subject Thermostability en_GB
dc.title Biochemical and structural characterisation of a thermophilic Aldo-Keto Reductase fromThermotoga maritima en_GB
dc.type Thesis or dissertation en_GB
dc.date.available 2009-03-24T14:27:45Z en_GB
dc.date.available 2011-01-26T08:33:25Z en_US
dc.date.available 2013-03-21T10:09:53Z
dc.contributor.advisor Littlechild, Jenny en_GB
dc.contributor.advisor Bycroft, Matthew en_GB
dc.publisher.department Biosciences en_GB
dc.type.degreetitle PhD in Biosciences en_GB
dc.type.qualificationlevel Doctoral en_GB
dc.type.qualificationname PhD en_GB


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