Antioxidant system within yeast peroxisome -: Biochemical and physiological characterization 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 residue. Pmp20 homologs containing the putative peroxisome targeting signal type 1 have also been identified in mammals and lower eukaryotes. However, the physiological function of these Pmp20 family proteins has been unclear. In this study, we investigated the biochemical and physiological functions of recombinant CbPmp20 protein in methanol-induced peroxisomes of C. boidinii using the PMP20-deleted strain of C. boidinii (pmp20 Delta strain). The His(6)-tagged CbPmp20 fusion protein was found to have glutathione peroxidase activity in vitro toward alkyl hydroperoxides and H2O2. Catalytic activity and dimerization of His(6)-CbPmp20 depended on the only cysteine residue corresponding 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 targeting signal type 1 sequence nor CbPmp20 haboring the C53S mutation retrieved the growth defect. Interestingly, the pmp20 Delta strain had a more severe growth defect than the cta1 Delta strain, which lacks catalase, another antioxidant enzyme within the peroxisome. During incubation of these strains in methanol medium, the cta1 Delta strain accumulated H2O2, whereas the pmp20 Delta strain did not. Therefore, it is speculated to be the main function of CbPmp20 is to decompose reactive oxygen species generated at peroxisomal membrane surface, e.g. lipid hydroperoxides, rather than to decompose H2O2. In addition, we detected a physiological level of reduced glutathione in peroxisomal fraction of C. boidinii. These results may indicate a physiological role for CbPmp20 as an antioxidant enzyme within peroxisomes rich in reactive oxygen species.