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Protein engineering and mechanistic studies of morphinone reductase

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posted on 2014-12-15, 10:32 authored by Hanan Latif Fahmi Messiha
The thesis describes mechanistic studies of the bacterial protein morphinone reductase (MR) using mutagenesis, kinetic and spectroscopic methods. The roles of seven active site residues were investigated. Cys-191 was initially predicted to be an active site acid; however, mutagenesis studies revealed that Cys-191 does not act in such role. His-186 and Asn-189 were shown to be involved in ligand binding. Mutation of His-186 to alanine has dramatic effects on the enzyme in the steady-state and in the oxidative half-reaction suggesting that His-186 could be an active site acid; however, pH dependence and NMR spectroscopic studies proved that His-186 could not play such role. No charge-transfer complex was observed between the H186A mutant and b-NADH in the reductive half-reaction. Mutation of Asn-189 affects enzyme kinetics and results in a slower rate of flavin oxidation. Faster rates for flavin reduction in the N189A mutant suggest that mutation of Asn-189 favours orientation of the flavin and b-NADH for hydride transfer. Neither Tyr-72 nor Tyr-356 is an active site acid. Mutation of Tyr-72 does not substantially affect enzyme kinetics. Mutations of Tyr-356 and Trp-106 had pronounced affects on enzyme kinetics, suggesting that each mutation alters the active site geometry. In the seven MR mutants studied, the flavin redox potentials are not altered compared to wild-type enzyme except for the T32A mutant where a 50 mV decrease is observed. Mutation of Thr-32 produced enhanced activity in the oxidative half-reaction and a slower activity in the reductive half-reaction effect of copper on MR was investigated. His-186 is shown to be involved in copper binding. 1-Nitrocyclohexene is a potential substrate for MR and the underlying mechanism requires further study.

History

Date of award

2004-01-01

Author affiliation

Biochemistry

Awarding institution

University of Leicester

Qualification level

  • Doctoral

Qualification name

  • PhD

Language

en

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