Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.626069
Title: Investigations of the arsenite oxidase from a model thermophile and characterisation of the enzyme from its mesophilic counterparts
Author: Heath, M. D.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2013
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Abstract:
The arsenite oxidase (Aio) catalyses the two-electron oxidation of arsenite to arsenate. The enzyme, purified and characterised from five mesophiles and one psychrotolerant bacterium, is a heterodimer with a large catalytic subunit (AioA) containing a molybdopterin guanine dinucleotide cofactor and 3Fe-4S center, and a small subunit (AioB) that contains a Rieske 2Fe-2S center. To understand the properties of a thermophilic arsenite oxidase, the enzyme from Thermus thermophilus str. HB8 was enriched and partially characterised. Because of the difficulties with enzyme purification and yield, the enzyme was heterologously expressed in Escherichia coli, purified and its properties compared to the native enzyme. The enzyme, also a heterodimer, was found to have maximal specific activity at 70oC. This was 10oC below and at least five-times less active than measurements taken from the study of the native enzyme. Comparisons with the heterologously expressed mesophilic arsenite oxidase from Alcaligenes faecalis were also made. The temperature profile of the A. faecalis enzyme was comparable to its mesophilic homologue from Rhizobium sp. NT-26, displaying an optimum temperature of 65oC. Mechanisms thought to contribute to the thermostability of the HB8 Aio were investigated using a homology model of the enzyme, which was constructed using the X-ray crystal structures of the arsenite oxidases from A. faecalis and NT-26. A major stabilising factor in the model appeared to be the substitution of charged/polar residues with non-polar/hydrophobic residues of the interior surface/core of the enzyme. Studies of the electron transfer pathway were conducted using an electrochemical approach and the heterologously expressed NT-26 arsenite oxidase and its native physiological electron acceptor, a cytochrome c552. The cytochrome was immobilised on an alkanethiol-modified gold electrode and used to mediate electrons between the enzyme and the electrode. This is the first electrochemical investigation of the arsenite oxidase in partnership with its native, physiological electron partner. A catalytic optimum of pH 6.5 was found and substrate Km of 277 µM. The “wiring” of the enzyme and its co-substrate (cytochrome c552) to an electrode in this way also highlighted its potential use in a wider context as a biosensor.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.626069  DOI: Not available
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