TY - JOUR T1 - The First Aspartic Acid of the DQxD Motif for Human UDP-Glucuronosyltransferase 1A10 Interacts with UDP-Glucuronic Acid during Catalysis JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 517 LP - 522 DO - 10.1124/dmd.107.016469 VL - 36 IS - 3 AU - Yan Xiong AU - Anne-Sisko Patana AU - Michael J. Miley AU - Agnieszka K. Zielinska AU - Stacie M. Bratton AU - Grover P. Miller AU - Adrian Goldman AU - Moshe Finel AU - Matt R. Redinbo AU - Anna Radominska-Pandya Y1 - 2008/03/01 UR - http://dmd.aspetjournals.org/content/36/3/517.abstract N2 - All UDP-glucuronosyltransferase enzymes (UGTs) share a common cofactor, UDP-glucuronic acid (UDP-GlcUA). The binding site for UDP-GlcUA is localized to the C-terminal domain of UGTs on the basis of amino acid sequence homology analysis and crystal structures of glycosyltransferases, including the C-terminal domain of human UGT2B7. We hypothesized that the 393DQMDNAK399 region of human UGT1A10 interacts with the glucuronic acid moiety of UDP-GlcUA. Using site-directed mutagenesis and enzymatic analysis, we demonstrated that the D393A mutation abolished the glucuronidation activity of UGT1A10 toward all substrates. The effects of the alanine mutation at Q394,D396, and K399 on glucuronidation activities were substrate-dependent. Previously, we examined the importance of these residues in UGT2B7. Although D393 (D398 in UGT2B7) is similarly critical for UDP-GlcUA binding in both enzymes, the effects of Q394 (Q399 in UGT2B7) to Ala mutation on activity were significant but different between UGT1A10 and UGT2B7. A model of the UDP-GlcUA binding site suggests that the contribution of other residues to cosubstrate binding may explain these differences between UGT1A10 and UGT2B7. We thus postulate that D393 is critical for the binding of glucuronic acid and that proximal residues, e.g., Q394 (Q399 in UGT2B7), play a subtle role in cosubstrate binding in UGT1A10 and UGT2B7. Hence, this study provides important new information needed for the identification and understanding of the binding sites of UGTs, a major step forward in elucidating their molecular mechanism. The American Society for Pharmacology and Experimental Therapeutics ER -