Antioxidant system within yeast peroxisome -: Biochemical and physiological characterization of CbPmp20 in the methylotrophic yeast Candida boidinii
Antioxidant system within yeast peroxisome -: Biochemical and physiological characterization of CbPmp20 in the methylotrophic yeast Candida boidinii
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DOI：
10.1074/jbc.m011661200
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发表时间：
2001-04-27
影响因子：
4.8
通讯作者：
Sakai, Y
中科院分区：
文献类型：
--
作者：
Horiguchi, H;Yurimoto, H;Sakai, Y
博伊丁假丝酵母Pmp 20（CbPmp 20）是一种与过氧化物酶体膜内侧结合的蛋白质，属于新近发现的一种含有半胱氨酸残基的抗氧化酶--过氧化物氧还蛋白家族。含有假定的过氧化物酶体靶向信号类型1的pmp 20同源物也已在哺乳动物和低等真核生物中鉴定。然而，这些Pmp 20家族蛋白的生理功能一直不清楚。本研究旨在探讨CbPmp 20重组蛋白在甲醇诱导的C. boidinii的PMP 20缺失菌株。boidinii（pmp 20 Delta菌株）。发现His（6）-标记的CbPmp 20融合蛋白在体外对烷基氢过氧化物和H2 O2具有谷胱甘肽过氧化物酶活性。His（6）-CbPmp 20的催化活性和二聚化依赖于对应于Cys（53）的唯一半胱氨酸残基。发现pmp 20 Delta菌株在甲醇作为碳源和能源上失去了生长能力。pmp 20 Delta生长缺陷被CbPmp 20挽救，但缺乏过氧化物酶体靶向信号类型1序列的CbPmp 20和具有C53 S突变的CbPmp 20都不能恢复生长缺陷。有趣的是，pmp 20 Delta菌株比cta 1 Delta菌株具有更严重的生长缺陷，cta 1 Delta菌株缺乏过氧化氢酶，过氧化物酶体内的另一种抗氧化酶。这些菌株在甲醇培养基中孵育期间，cta 1 Delta菌株积累H2 O2，而pmp 20 Delta菌株没有。因此，推测CbPmp 20的主要功能是分解在过氧化物酶体膜表面产生的活性氧物质，例如脂质氢过氧化物，而不是分解H2 O2。此外，我们还在C.博伊丁尼。这些结果可能表明CbPmp 20作为过氧化物酶体内富含活性氧的抗氧化酶的生理作用。
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.