L-pipecolic acid oxidase, a human enzyme essential for the degradation of L-pipecolic acid, is most similar to the monomeric sarcosine oxidases
L-pipecolic acid oxidase, a human enzyme essential for the degradation of L-pipecolic acid, is most similar to the monomeric sarcosine oxidases
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DOI：
10.1042/0264-6021:3450487
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发表时间：
2000-02-01
影响因子：
4.1
通讯作者：
Mihalik, SJ
中科院分区：
文献类型：
--
作者：
Dodt, G;Kim, DG;Mihalik, SJ
l -胡椒酸氧化酶活性在过氧化物酶体生物发生障碍（PBDs）患者中缺乏。由于其在这些疾病中的作用（如果有的话）尚不清楚，我们克隆了相关的人类基因并表达了其蛋白产物。利用从猴l -果酸氧化酶纯化得到的氨基酸序列，利用反向遗传学方法克隆了该cDNA。通过常规文库筛选和cDNA末端的5'快速扩增获得完整的cDNA，包含1170个碱基的开放阅读框，可翻译成390个残基的蛋白。翻译后的蛋白以序列AHL终止，这是一个过氧化物酶体靶向信号1。间接免疫荧光研究表明，该蛋白产物在人成纤维细胞中以与过氧化物酶过氧化氢酶共定位的点状模式表达。经BLAST序列分析，与Bacillus sp. NS-129单体肌氨酸氧化酶同源性为31%，相似度为53%，与所有的肌氨酸氧化酶和脱氢酶相似，与过氧化物酶体d -氨基酸氧化酶无相似性。重组酶能同时氧化l -胡椒果酸和肌氨酸。然而，缺乏该酶活性的PBD患者仅积累l -胡椒果酸，这表明在人体体内，该酶主要参与l -胡椒果酸的降解。
L-Pipecolic acid oxidase activity is deficient in patients with peroxisome biogenesis disorders (PBDs). Because its role, if any, in these disorders is unknown, we cloned the associated human gene and expressed its protein product. The cDNA was cloned with the use of a reverse genetics approach based on the amino acid sequence obtained from purified L-pipecolic acid oxidase from monkey. The complete cDNA, obtained by conventional library screening and 5' rapid amplification of cDNA ends, encompassed an open reading frame of 1170 bases, translating to a 390-residue protein. The translated protein terminated with the sequence AHL, a peroxisomal targeting signal 1. Indirect immunofluorescence studies showed that the protein product was expressed in human fibroblasts in a punctate pattern that co-localized with the peroxisomal enzyme catalase. A BLAST search with the amino acid sequence showed 31% identity and 53% similarity with Bacillus sp. NS-129 monomeric sarcosine oxidase, as well as similarity to all sarcosine oxidases and dehydrogenases, No similarity was found to the peroxisomal D-amino acid oxidases. The recombinant enzyme oxidized both L-pipecolic acid and sarcosine. However, PBD patients who lack the enzyme activity accumulate only L-pipecolic acid, suggesting that in humans in vivo, this enzyme is involved mainly in the degradation of L-pipecolic acid.