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The First Aspartic Acid of the DQxD Motif for Human UDP-Glucuronosyltransferase 1A10 Interacts with UDP-Glucuronic Acid during Catalysis

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas (Y.X., A.K.Z., S.M.B., G.P.M., A.R.-P.); Structural Biology and Biophysics, Institute of Biotechnology (A.-S.P., A.G.), and Neuroscience Center (A.G.), University of Helsinki, Helsinki, Finland; Departments of Chemistry (M.J.M.) and Biochemistry and Biophysics (M.R.R.), Program in Molecular Biology and Biotechnology (M.R.R.), and the Lineberger Comprehensive Cancer Center (M.R.R.), University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; and Drug Discovery and Development Technology Center, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland (M.F.)

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.