Abstract

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.