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-glu curonosyltransferase (UGT) enzymes. Previously, two cDNA clones, UGT2B15 an d UGT2B17, which encode UGT enzymes capable of glucuronidating C-19 steroid s, were isolated and characterized. These proteins are 95% identical in pri mary structure; however, UGT2B17 is capable of conjugating C-19 steroid mol ecules at both the 3 alpha and 17 beta-OH positions, whereas UGT2B15 is onl y active at the 17 beta-OH position. To identify the amino acid residue(s) which may account for this difference in substrate specificity, a comprehen sive study on the role of 15 residues which differ between UGT2B15 and UGT2 B17 was performed by site-directed mutagenesis. The stable expression of UG T2B17 mutant proteins into HK293 cells demonstrated that the mutation of is oleucine 125, valine 181 and valine 455 to the residues found in UGT2B15 di d not alter enzyme activity nor substrate specificity. Furthermore, mutatio n of the variant residues in UGT2B15 (serine 124, asparagine 125, phenylala nine 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 t he ability of UGT2B17 to conjugate androsterone at the 3 alpha position, bu t still retained activity for dihydrotestosterone and 5 alpha-androstane-3 alpha,17 beta-diol, which have an OH-group at the 17 beta position. Interes tingly, mutation of tyrosine 121 in UGT2B15 to a serine abolished activity for C-19 steroids. It is suggested that the serine residue at position 121 in UGT2B17 is required for activity towards the 3 alpha and not for the 17 beta position of C-19 steroids, whereas the tyrosine 121 in UGT2B15 is nece ssary for UGT activity. Despite the high homology between UGT2B15 and UGT2B 17, it is apparent that different amino acid residues in the two proteins a re required to confer conjugation of C-19 steroid molecules. (C) 1999 Acade mic Press.