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The Saccharomyces cerevisiae quinone oxidoreductase Lot6p: stability, inhibition and cooperativity.
Megarity, Clare F; Looi, Hong Keat; Timson, David J.
Afiliação
  • Megarity CF; School of Biological Sciences, Medical Biology Centre, Queen's University Belfast, Belfast, UK.
FEMS Yeast Res ; 14(5): 797-807, 2014 Aug.
Article em En | MEDLINE | ID: mdl-24866129
ABSTRACT
Lot6p (EC 1.5.1.39; Ylr011wp) is the sole quinone oxidoreductase in the budding yeast, Saccharomyces cerevisiae. Using hexahistidine tagged, recombinant Lot6p, we determined the steady-state enzyme kinetic parameters with both NADH and NADPH as electron donors; no cooperativity was observed with these substrates. The NQO1 inhibitor curcumin, the NQO2 inhibitor resveratrol, the bacterial nitroreductase inhibitor nicotinamide and the phosphate mimic vanadate all stabilise the enzyme towards thermal denaturation as judged by differential scanning fluorimetry. All except vanadate have no observable effect on the chemical cross-linking of the two subunits of the Lot6p dimer. These compounds all inhibit Lot6p's oxidoreductase activity, and all except nicotinamide exhibit negative cooperativity. Molecular modelling suggests that curcumin, resveratrol and nicotinamide all bind over the isoalloxazine ring of the FMN cofactor in Lot6p. Resveratrol was predicted to contact an α-helix that links the two active sites. Mutation of Gly-142 (which forms part of this helix) to serine does not greatly affect the thermal stability of the enzyme. However, this variant shows less cooperativity towards resveratrol than the wild type. This suggests a plausible hypothesis for the transmission of information between the subunits and, thus, the molecular mechanism of negative cooperativity in Lot6p.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Quinonas / Saccharomyces cerevisiae / Proteínas de Saccharomyces cerevisiae / FMN Redutase Tipo de estudo: Prognostic_studies Idioma: En Revista: FEMS Yeast Res Assunto da revista: MICROBIOLOGIA Ano de publicação: 2014 Tipo de documento: Article País de afiliação: Reino Unido

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Quinonas / Saccharomyces cerevisiae / Proteínas de Saccharomyces cerevisiae / FMN Redutase Tipo de estudo: Prognostic_studies Idioma: En Revista: FEMS Yeast Res Assunto da revista: MICROBIOLOGIA Ano de publicação: 2014 Tipo de documento: Article País de afiliação: Reino Unido