Received for publication May 3, 2007.
Revised November 16, 2007.
Accepted for publication November 26, 2007.

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

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 based on 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. While D³⁹³ (D³⁹⁸ in UGT2B7) is similarly critical for UDP-GlcUA binding in both enzymes, the effects of the Q³⁹⁴ (Q³⁹⁹ in UGT2B7) to Ala mutation on activity was significant but different between UGT1A10 and UGT2B7. A model of the UDP-GlcUA binding site suggests that the contribution of other residues to co-substrate 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 co-substrate 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.

Key words: computer modeling and simulation, phase II drug metabolism, site-directed mutagenesis, structure-activity relationships, UDP glucuronyltransferases

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