Lot6p from Saccharomyces cerevisiae is a FMN-dependent reductase with a potential role in quinone detoxification.
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 microm 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 Deltalot6 and Lot6p overexpressing strains, suggesting that Lot6p may play a role in managing oxidative stress in yeast.
Sollner
Sonja
S
Institute of Biochemistry, Graz University of Technology, Petersgasse 12/II, A-8010 Graz, Austria.
Nebauer
Ruth
R
Ehammer
Heidemarie
H
Prem
Anna
A
Deller
Sigrid
S
Palfey
Bruce A
BA
Daum
Günther
G
Macheroux
Peter
P
D000693:Anaerobiosis; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D001728:Dicumarol / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D038181:FMN Reductase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008658:Inactivation, Metabolic; D007211:Indoles; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D011809:Quinones / Q000633:toxicity*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013347:Subcellular Fractions
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007211:Indoles; D011809:Quinones; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D009243:NAD; D049452:Green Fluorescent Proteins; C007293:DAPI; D009249:NADP; D001728:Dicumarol; D038181:FMN Reductase; C490376:LOT6 protein, S cerevisiae