title: The Saccharomyces cerevisiae quinone oxidoreductase Lot6p: stability, inhibition and cooperativity.
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.
authors.affiliation: School of Biological Sciences, Medical Biology Centre, Queen's University Belfast, Belfast, UK.
authors.forename: Clare F
authors.initials: CF
authors.lastname: Megarity
authors.forename: Hong Keat
authors.initials: HK
authors.lastname: Looi
authors.forename: David J
authors.initials: DJ
authors.lastname: Timson
mesh_terms: D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability / Q000528:radiation effects; D038181:FMN Reductase / Q000737:chemistry / Q000378:metabolism*; D005470:Fluorometry; D007700:Kinetics; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011809:Quinones / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D011809:Quinones; D029701:Saccharomyces cerevisiae Proteins; D009243:NAD; D009249:NADP; D038181:FMN Reductase; C490376:LOT6 protein, S cerevisiae
keywords: FMN-containing enzyme; negative cooperativity; nitroreductase; quinone oxidoreductase; resveratrol