Abstract

Human UDP-glucuronosyltransferase (UGT)1A9 is one of the major isoforms in liver and extrahepatic tissues, catalyzing the glucuronidation of a variety of drugs, dietary constituents, steroids, fatty acids, and bile acids. UGT1A9 shows high amino acid homology with UGT1A7, UGT1A8, and UGT1A10 with overlapping substrate specificity. However, the affinities for substrates are different among them. Amino acid alignment analysis revealed that 14 amino acids, Cys3, Arg42, Lys91, Ala92, Tyr106, Gly111, Tyr113, Asp115, Asn152, Leu173, Leu219, His221, Arg222, and Glu241, are unique to UGT1A9 compared with UGT1A7, UGT1A8, and UGT1A10. In this study, we constructed expression systems in human embryonic kidney 293 cells for seven mutants (Mut) UGT1A9, Mut 1 (R42Q), Mut 2 (K91M, A92D), Mut 3 (Y106F, G111S, D115G), Mut 4 (N152A), Mut 5 (L173A), Mut 6 (L219F, H221Q, R222Y), and Mut 7 (E241A), in which the amino acids were substituted to those of UGT1A8. Using these mutants, the effects of the amino acid changes on the activities of 4-methylumbelliferone (4-MU), p-nitrophenol (p-NP), and 3-hydroxydesloratadine glucuronidations were investigated. For 4-MU and p-NP O-glucuronidations, Mut 1 and Mut 4 exhibited higher K_m values and Mut 3 and Mut 4 exhibited higher V_max values compared with wild-type UGT1A9. It is interesting to note that only Mut 4 was active toward 3-hydroxydesloratadine O-glucuronidation that is specific for UGT1A8. The findings reveal that the residues Arg42 and Asn152 may have a large contribution to the difference in the substrate specificity with that of UGT1A8, although all of the unique amino acids of UGT1A9 would be collectively involved in the catalytic property.