Crystal Structure of the Cofactor-Binding Domain of the Human Phase II Drug-Metabolism Enzyme UDP-Glucuronosyltransferase 2B7

Abstract

SummaryHuman UDP-glucuronosyltransferases (UGT) are the dominant phase II conjugative drug metabolism enzymes that also play a central role in the processing of a range of endobiotic compounds. UGTs catalyze the covalent addition of glucuronic acid sugar moieties to a host of therapeutics and environmental toxins, as well as to a variety of endogenous steroids and other signaling molecules. We report the 1.8 Å resolution apo crystal structure of the UDP-glucuronic acid binding domain of human UGT isoform 2B7 (UGT2B7), which catalyzes the conjugative elimination of opioid, antiviral, and anticancer drugs. This is the first crystal structure of any region of a mammalian UGT drug metabolism enzyme. Designed UGT2B7 mutants at residues predicted to interact with the UDP-glucuronic acid cofactor exhibited significantly impaired catalytic activity, with maximum effects observed for amino acids closest to the glucuronic acid sugar transferred to the acceptor molecule. Homology modeling of UGT2B7 with related plant flavonoid glucosyltransferases indicate that human UGTs share a common catalytic mechanism. Point mutations at predicted catalytic residues in UGT2B7 abrogated activity, strongly suggesting that human UGTs also utilize a serine hydrolase-like catalytic mechanism to facilitate glucuronic acid transfer.

Identification of Aspartic Acid and Histidine Residues Mediating the Reaction Mechanism and the Substrate Specificity of the Human UDP-glucuronosyltransferases 1A

“…By analogy with the reaction mechanism that has been described for crystallized glycosyltransferases, we postulated that His-370 would stabilize the leaving UDP group during catalysis by neutralizing the negative charge of the pyrophosphate moiety (34). This assumption was recently supported by structure analysis of the C-terminal domain of UGT2B7 (residues 285-451) predicting that His-374 residue (His-370 in UGT1A6) interacts with the ␤-phosphate of the donor substrate UDP-GlcUA (14).…”

“…Our findings are consistent with Asp-394 and Asp-397 being important for the interaction of UGT1A6 with UDP-GlcUA. This is supported by structural analysis of the C-terminal domain of UGT2B7 that predicted that Asp-398 (Asp-394 in UGT1A6) interacts with GlcUA moiety of the co-substrate (14).…”

“…Briefly, incubation in Eppendorf tubes (total volume 40 l) consisted of 50 g of microsomal proteins in 100 mM Tris-HCl buffer (pH 7.4), 10 mM MgCl 2 containing 0.1 mM UDP-GlcUA and 0.1 Ci UDP-[U- 14 C]glucuronic acid. The reaction was started by the addition of substrate (0.5 mM, final concentration) dissolved in 2 l of dimethyl sulfoxide.…”

“…Recently, the structure of the cofactor binding domain of UGT2B7 has been solved, and several amino acids interacting with the co-substrate (UDP-GlcUA) have been identified (14). However, the lack of information on the threedimensional structure of the substrate binding domain and of the full-length protein due to difficulties in crystallizing these membrane proteins has impaired a better understanding of the active site organization and of the glucuronidation process.…”

Topological aspects of oligomeric UDP-glucuronosyltransferases in endoplasmic reticulum membranes: Advances and open questions

“…The crystal structure of the UDPGA-binding Cterminal half of UGT2B7 has recently been determined at 1.8-Å resolution. Mutants at residues predicted to interact with UDPGA exhibited impaired catalytic activity, and mutants at predicted aglycone binding sites abrogated UGT activity [11]. In addition, an internal signal sequence has been identified embedding part of the N-terminal half of UGTs in the ER membrane, a feature which may facilitate access of lipophilic aglycones to the active site [12].…”