TheSaccharomyces cerevisiaequinone 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.

Dicoumarol: A Drug which Hits at Least Two Very Different Targets in Vitamin K Metabolism

“…However, it is interesting to note that similar effects have also been observed in the related human enzyme NRH-quinone oxidoreductase 2 (NQO2) and the budding yeast quinone oxidoreductase Lot6p [88,89]. The crystal structure of NQO1 bound to dicoumarol shows that the ligand binds to both active sites, lying above the isoalloxazine ring of the FAD cofactors [90].…”

A novel cytosolic NADH:quinone oxidoreductase from Methanothermobacter marburgensis

“…In addition to bacterial MdaBs, Mm NQO displays structural similarities to the FMN-dependent NAD(P)H:quinone oxidoreductase Saccharomyces cerevisiae Lot6p (former YLR011wp; PDB code 1T0I [ 46 ]). Sc Lot6p has been shown to act as an inducer of apoptosis [ 47 ] and is the only known quinone reductase in budding yeast [ 48 ]. The structure-based sequence identity between Mm NQO and S cLot6p is 13.8%, which is slightly higher than the pairwise identity of 13.3% between Mm NQO and Ec MdaB.…”

Escherichia coli Modulator of Drug Activity B (MdaB) Has Different Enzymological Properties to Eukaryote Quinone Oxidoreductases

“…The vector inserts a sequence which codes for residues, MAHHHHHHVDDDDK at the 5' end of the gene enabling purification of the recombinant proteins by Ni 2 + chromatography (His-Select, Sigma, UK); expression and purification were carried out as previously described for budding yeast Lot6p. [20] Protein concentrations were estimated by the method of Bradford using BSA as a control. [35] Chemical crosslinking was carried out as previously described.…”

“…Differential scanning fluorimetry was carried out as previously described. [20] An initial titration was carried out to identify an enzyme concentration which gave an optimum fluorescent signal. Enzymes were diluted in 50 mM HEPES, pH 7.33 to final concentrations in the range 0.25 μM to 5 μM dimer.…”

“…[11] Human NAD(P)H quinone oxidoreductase 1 (NQO1), human NRH quinone oxidoreductase 2 (NQO2) and budding yeast Lot6p all exhibit negative cooperativity towards inhibitors such as dicoumarol and resveratrol. [19][20][21][22] Negative cooperativity is a decrease in affinity when sequential molecules of ligand bind. [23,24] Binding of the first molecule causes a conformational change which is propagated through the protein and causes the effect elsewhere.…”