Site-Directed Mutagenesis of Dimethyl Sulfoxide Reductase from Rhodobacter capsulatus: Characterization of a Y114 F Mutant

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• 作者：Justin P. Ridge ; Kondo-Francois Aguey-Zinsou ; Paul V. Bernhardt ; Ian M. Brereton ; Graeme R. Hanson ; and Alastair G. McEwan
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：December 31, 2002
• 年：2002
• 卷：41
• 期：52
• 页码：15762 - 15769
• 全文大小：94K
• 年卷期：v.41,no.52(December 31, 2002)
• ISSN：1520-4995

文摘

A system for expressing site-directed mutants of the molybdenum enzyme dimethyl sulfoxidereductase from Rhodobacter capsulatus in the natural host was constructed. This system was used togenerate and express dimethyl sulfoxide reductase with a Y114F mutation. The Y114F mutant had anincreased k_cat and increased K_m toward both dimethyl sulfoxide and trimethylamine N-oxide compared tothe native enzyme, and the value of k_cat/K_m was lower for both substrates in the mutant enzyme. TheY114F mutant, as isolated, was able to oxidize dimethyl sulfide with phenazine ethosulfate as the electronacceptor but with a lower k_cat than that of the native enzyme. The pH optimum of dimethyl sulfide:acceptor oxidoreductase activity in the Y114F mutant was shown to be shifted by +1 pH unit comparedto the native enzyme. The Y114F mutant did not form a pink complex with dimethyl sulfide, which ischaracteristic of the native enzyme. The mutant enzyme showed a large increase in the K_d for DMS.Direct electrochemistry showed that the Mo(V)/Mo(IV) couple was unaffected by the Y114F mutant, butthe midpoint potential of the Mo(VI)/Mo(V) couple was raised by about 50 mV. These data confirm thatthe Y114 residue plays a critical role in oxidation-reduction processes at the molybdenum active siteand in oxygen atom transfer associated with sulfoxide reduction.