Antioxidant system within yeast peroxisome - Biochemical and physiologicalcharacterization of CbPmp20 in the methylotrophic yeast Candida boidinii

Candida boidinii Pmp20 (CbPmp20), a protein associated with the inner side of peroxisomal membrane, belongs to a recently identified protein family of antioxidant enzymes, the peroxiredoxins, which contain one cysteine residu e. Pmp20 homologs containing the putative peroxisome targeting signal type 1 have also been identified in mammals and lower eukaryotes. However, the p hysiological function of these Pmp20 family proteins has been unclear. In t his study, we investigated the biochemical and physiological functions of r ecombinant CbPmp20 protein in methanol-induced peroxisomes of C. boidinii u sing the PMP20-deleted strain of C. boidinii (pmp20 Delta strain). The His( 6)-tagged CbPmp20 fusion protein was found to have glutathione peroxidase a ctivity in vitro toward alkyl hydroperoxides and H2O2. Catalytic activity a nd dimerization of His(6)-CbPmp20 depended on the only cysteine residue cor responding to Cys(53). The pmp20 Delta strain was found to have lost growth ability on methanol as a carbon and energy source. The pmp20 Delta growth defect was rescued by CbPmp20, but neither CbPmp20 lacking the peroxisome t argeting signal type 1 sequence nor CbPmp20 haboring the C53S mutation retr ieved the growth defect. Interestingly, the pmp20 Delta strain had a more s evere growth defect than the cta1 Delta strain, which lacks catalase, anoth er antioxidant enzyme within the peroxisome. During incubation of these str ains in methanol medium, the cta1 Delta strain accumulated H2O2, whereas th e pmp20 Delta strain did not. Therefore, it is speculated to be the main fu nction of CbPmp20 is to decompose reactive oxygen species generated at pero xisomal membrane surface, e.g. lipid hydroperoxides, rather than to decompo se H2O2. In addition, we detected a physiological level of reduced glutathi one in peroxisomal fraction of C. boidinii. These results may indicate a ph ysiological role for CbPmp20 as an antioxidant enzyme within peroxisomes ri ch in reactive oxygen species.