Alteration of human UDP-glucuronosyltransferase UGT2B17 regio-specificity by a single amino acid substitution

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 C19 steroids, were isolated and characterized. These proteins are 95 % identical in primary structure; however, UGT2B17 is capable of conjugating C19 steroid molecules at both the 3α and 17β-OH positions, whereas UGT2B15 is only active at the 17β-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 3α position, but still retained activity for dihydrotestosterone and 5α-androstane-3α,17β-diol, which have an OH-group at the 17β position. Interestingly, mutation of tyrosine 121 in UGT2B15 to a serine abolished activity for C19 steroids. It is suggested that the serine residue at position 121 in UGT2B17 is required for activity towards the 3α and not for the 17β position of C19 steroids, 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 C19 steroid molecules.