Dubois S G, Beaulieu M, Lévesque E, Hum D W, Bélanger A
Medical Research Council Group in Molecular Endocrinology, CHUL Research Center, Laval University, Québec, G1V 4G2, Canada.
J Mol Biol. 1999 May 28;289(1):29-39. doi: 10.1006/jmbi.1999.2735.
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.
类固醇激素的葡萄糖醛酸化由尿苷二磷酸葡萄糖醛酸基转移酶（UGT）家族的酶催化。此前，已分离并鉴定了两个cDNA克隆，即UGT2B15和UGT2B17，它们编码能够使C19类固醇葡萄糖醛酸化的UGT酶。这些蛋白质的一级结构有95%相同；然而，UGT2B17能够在3α和17β - OH位置结合C19类固醇分子，而UGT2B15仅在17β - OH位置有活性。为了确定可能导致这种底物特异性差异的氨基酸残基，通过定点诱变对UGT2B15和UGT2B17之间不同的15个残基的作用进行了全面研究。将UGT2B17突变蛋白稳定表达于HK293细胞中表明，将异亮氨酸125、缬氨酸181和缬氨酸455突变为UGT2B15中的残基不会改变酶活性和底物特异性。此外，将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中的氨基酸残基也不会改变酶活性和底物特异性。然而，将UGT2B17中121位的丝氨酸残基突变为UGT2B15中的酪氨酸，消除了UGT2B17在3α位置结合雄甾酮的能力，但仍保留了对在17β位置有OH基团的双氢睾酮和5α - 雄甾烷 - 3α, 17β - 二醇的活性。有趣的是，将UGT2B15中的酪氨酸121突变为丝氨酸消除了对C19类固醇的活性。这表明UGT2B17中121位的丝氨酸残基是对C19类固醇3α位置活性所必需的，而非17β位置，而UGT2B15中的酪氨酸121对UGT活性是必需的。尽管UGT2B15和UGT2B17之间有高度同源性，但显然两种蛋白质中不同的氨基酸残基是赋予C19类固醇分子结合能力所必需的。
