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首页> 外文期刊>Journal of Molecular Biology >Alteration of human UDP-glucuronosyltransferase UGT2B17 regio-specificity by a single amino acid substitution.
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Alteration of human UDP-glucuronosyltransferase UGT2B17 regio-specificity by a single amino acid substitution.

机译：通过单个氨基酸取代来改变人UDP-葡萄糖醛酸转移酶UGT2B17的区域特异性。

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The glucuronidation of steroid hormones is catalyzed by a family of UDP-glucuronosyltransferase (UGT) enzymes. Previously, two cDNA clones, UGT2B15 and UGT2B17, which encode UGT enzymes capable of glucuronidating C19steroids, were isolated and characterized. These proteins are 95% identical in primary structure; however, UGT2B17 is capable of conjugating C19steroid molecules at both the 3alpha and 17beta-OH positions, whereas UGT2B15 is only active at the 17beta-OH position. To identify the amino acid residue(s) which may account for this difference in substrate specificity, a comprehensive study on the role of 15 residues which differ between UGT2B15 and UGT2B17 was performed by site-directed mutagenesis. The stable expression of UGT2B17 mutant proteins into HK293 cells demonstrated that the mutation of isoleucine 125, valine 181 and valine 455 to the residues found in UGT2B15 did not alter enzyme activity nor substrate specificity. Furthermore, mutation of the variant residues in UGT2B15 (serine 124, asparagine 125, phenylalanine 165) to the amino acid residues found in UGT2B17 did not alter enzyme activity nor substrate specificity. However, mutation of the serine residue at position 121 of UGT2B17 to a tyrosine, as found in UGT2B15, abolished the ability of UGT2B17 to conjugate androsterone at the 3alpha position, but still retained activity for dihydrotestosterone and 5alpha-androstane-3alpha, 17beta-diol, which have an OH-group at the 17beta position. Interestingly, mutation of tyrosine 121 in UGT2B15 to a serine abolished activity for C19steroids. It is suggested that the serine residue at position 121 in UGT2B17 is required for activity towards the 3alpha and not for the 17beta position of C19steroids, whereas the tyrosine 121 in UGT2B15 is necessary for UGT activity. Despite the high homology between UGT2B15 and UGT2B17, it is apparent that different amino acid residues in the two proteins are required to confer conjugation of C19steroid molecules. Copyright 1999 Academic Press.
机译：类固醇激素的葡糖醛酸化作用是由UDP-葡糖醛酸糖基转移酶（UGT）酶催化的。以前，已分离并鉴定了两个cDNA克隆UGT2B15和UGT2B17，它们编码能够葡萄糖醛酸化C19类固醇的UGT酶。这些蛋白质的一级结构具有95％的一致性;但是，UGT2B17能够在3alpha和17beta-OH位置偶联C19类固醇分子，而UGT2B15仅在17beta-OH位置具有活性。为了鉴定可解释底物特异性差异的氨基酸残基，通过定点诱变对UGT2B15和UGT2B17之间的15个残基的作用进行了全面研究。 UGT2B17突变蛋白在HK293细胞中的稳定表达表明，异亮氨酸125，缬氨酸181和缬氨酸455突变为UGT2B15中发现的残基不会改变酶活性或底物特异性。此外，UGT2B15中的变异残基（丝氨酸124，天冬酰胺125，苯丙氨酸165）突变为UGT2B17中发现的氨基酸残基不会改变酶的活性或底物特异性。但是，如在UGT2B15中发现的，将UGT2B17的121位丝氨酸残基突变为酪氨酸，取消了UGT2B17在3alpha位置缀合雄甾酮的能力，但仍保留了对二氢睾酮和5alpha-androstane-3alpha，17beta-二醇的活性，在17beta位置有一个OH-基团。有趣的是，UGT2B15中的酪氨酸121突变为丝氨酸，消除了C19类固醇的活性。有人建议，UGT2B17中121位的丝氨酸残基对于C19类固醇的3alpha活性是必需的，而不是C19类固醇的17beta位，而UGT2B15中的酪氨酸121是UGT活性所必需的。尽管UGT2B15和UGT2B17之间具有高度同源性，但很明显，需要两种蛋白质中的不同氨基酸残基来赋予C19类固醇分子缀合。版权所有1999，学术出版社。

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