Lot6p from Saccharomyces cerevisiae is a FMN‐dependent reductase with a potential role in quinone detoxification

Abstract

Several cellular oxidoreductases are involved in the metabolism of quinone compounds by catalyzing oneor two electron reductions of quinones. The cytotoxic effects of quinones are commonly thought to arise from their one-electron reduction, for example, by cytochrome P450 reductases, resulting in oxidative cycling of deleterious oxygen species.In contrast to other cellular reductases, NAD(P)H:quinone acceptor oxidoreductases (QRs) catalyze strict two-electron reductions and thereby afford protection against cytotoxic and neoplastic effects of electrophilic quinones [1]. For example, it has been shown that QR-null mice are more susceptible to quinone toxicity than their wild-type counterparts [2]. Furthermore, QRs are found in many solid tumors at increased levels and therefore can be used to target the tumor cells through bioreductive activation of quinone-based pro-drugs such as mitomycins [3].Cytosolic mammalian QRs have been crystallized from human (quinone reductase type 1 and 2) [4,5], rat [6] and mouse [4]. All of them are homodimers, containing FAD as a cofactor and exhibiting a flavodoxin-like fold that is characterized by twisted, parallel b-strands at the centre flanked by helices on both sides. The FAD cofactor is bound to the enzyme in the same manner, with the isoalloxazine ring system of the molecule accessible to the solvent and the ribityl phosphate and AMP moiety buried in the protein. NAD(P)H:quinone acceptor oxidoreductases are flavoenzymes expressed in the cytoplasm of many tissues and afford protection against the cytotoxic effects of electrophilic quinones by catalyzing a strict two-electron reduction. Such enzymes have been reported from several mammalian sources, e.g. human, mouse and rat, and from plant species. Here, we report identification of Lot6p (YLR011wp), the first soluble quinone reductase from the unicellular model organism Saccharomyces cerevisiae. Localization studies using an antibody raised against Lot6p as well as microscopic inspection of Lot6p-GFP demonstrated accumulation of the enzyme in the cytosol of yeast cells. Despite sharing only 23% similarity to type 1 human quinone reductase, Lot6p possesses biochemical properties that are similar to its human counterpart. The enzyme catalyzes a two-electron reduction of a series of natural and artificial quinone substrates at the expense of either NADH or NADPH. The kinetic mechanism follows a ping-pong bi-bi reaction scheme, with K M values of 1.6-11 lm for various quinones. Dicoumarol and Cibacron Marine, two well-known inhibitors of the quinone reductase family, bind to Lot6p and inhibit its activity. In vivo experiments demonstrate that the enzymatic activity of Lot6p is consistent with the phenotype of both Dlot6 and Lot6p overexpressing strains, suggesting that Lot6p may play a role in managing oxidative stress in yeast.Abbreviations duroquinone, 2,3,5,6-tetramethyl-1,4-benzoquinone; GFP, green fluorescent protein; Ni-NTA, nickel-nitrilotriacetic acid agarose; QR, quinone reductase.

Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases

“…Enzymes able to reduce an azo bond have been found in a number of species including Escherichia coli (Nakanishi et al, 2001), Pseudomonas aeruginosa (Wang et al, 2007), Enterococcus faecalis (Chen et al, 2004), Sinorhizobium meliloti (Ye et al, 2007), Saccharomyces cerevisiae (Sollner et al, 2007) and humans (Cui et al, 1995). These enzymes are flavin dependent oxidoreductases.…”

E3 Ligase STUB1/CHIP Regulates NAD(P)H:Quinone Oxidoreductase 1 (NQO1) Accumulation in Aged Brain, a Process Impaired in Certain Alzheimer Disease Patients

“…NQO1 was shown to be associated with the 20S proteasome preventing the degradation of various key regulatory proteins and enzymes such as p53, p73, p33, and c-Fos (4 -6). The association of NQO1 with the 20S is conserved over evolution, and it was recently shown in yeast that Lot6, the NQO1 ortholog (7), is also associated with the 20S proteasome (8).…”

Functional Annotation of a Presumed Nitronate Monoxygenase Reveals a New Class of NADH:Quinone Reductases

“…As mentioned earlier, very few FMN-dependent quinone reductases specific for NADH have been identified. Other FMN-dependent quinone reductases, which however utilize both NADH and NADPH, have also been identified in bacteria, fungi, and plants, including Methanothermobacter marbergensis NQO (45), Lot6p from Saccharomyces cerevisiae (46), YhdA (47), FQR1 (48), and NQR (49), among others. It is worth noting that other flavin-dependent enzymes with known functions have been shown to exhibit NAD(P)H:quinone reductase activity, for example bacterial nitroreductases and glucose oxidase (50).…”