Mapping the UDP-glucuronic Acid Binding Site in UDP-glucuronosyltransferase-1A10 by Homology-Based Modeling: Confirmation with Biochemical Evidence

Abstract

The UDP-glucuronosyltransferase (UGT) isozyme system is critical for protecting the body against endogenous and exogenous chemicals by linking glucuronic acid donated by UDP-glucuronic acid to a lipophilic acceptor substrate. UGTs convert metabolites, dietary constituents and environmental toxicants to highly excretable glucuronides. Because of difficulties associated with purifying endoplasmic reticulum-bound UGTs for structural studies, we carried out homology-based computer modeling to aid analysis. The search found structural homology in Escherichia coli UDP-galactose 4-epimerase. Consistent with predicted similarities involving the common UDP-moiety in substrates, UDP-glucose and UDP-hexanol amine caused competitive inhibition by Lineweaver-Burk plots. Among predicted binding sites N292, K314, K315 and K404 in UGT1A10, two informative sets of mutants K314R/Q/A/E /G and K404R/E had null activities or 2.7-fold higher/50% less activity, respectively. Scatchard analysis of binding data of affinity-ligand, 5-azido-uridine-[β-32 P]-diphosphoglucuronic acid, to purified UGT1A10-His or UGT1A7-His revealed high and low affinity binding sites. 2-Nitro 5-thiocyanobenzoic acid-digested UGT1A10-His bound with radiolabeled affinity-ligand revealed an 11.3-and 14.3-kDa peptide associated with K314 and K404, respectively, in a discontinuous SDS-PAGE system. Similar treatment of 1A10His-K314A bound with the ligand lacked both peptides; 1A10-HisK404R-and 1A10-HisK404E showed 1.3-fold greater-and 50% lesslabel in the 14.3-kDa peptide, respectively, compared to 1A10-His without affecting the 11.3-kDa peptide. Scatchard analysis of binding data of affinity-ligand to 1A10His-K404R and -K404E showed a 6-fold reduction and a large increase in Kd, respectively. Our results indicate: K314 and K404 are required UDP-glcA binding sites in 1A10, that K404 controls activity and high affinity sites and that K314 and K404 are strictly conserved in 70 aligned UGTs, except for S321--equivalent to K314--in UGT2B15 and 2B17 and I321 in the inactive UGT8, which suggests UGT2B15 and 2B17 contain suboptimal activity. Hence our data strongly support UDPglcA binding to K314 and K404 in UGT1A10.UGT isozymes carry out the critical role of detoxifying innumerable structurally diverse chemicals encountered on a regular basis, including toxic metabolites, dietary constituents, environmental carcinogens and, inadvertently, therapeutic agents. All UGT isozymes, which are distributed primarily in liver, kidney and gastrointestinal tract, transfer glucuronic acid † This research was supported by the Intramural Research Program of the Eunice Kennedy Shriver NICHD/NIH. ‡To whom correspondence should be addressed: E-mail: owensi@mail.nih.gov. SUPPORTING INFORMATION AVAILABLE SeqFold alignment of UGT1A10 to 1XEL (Fig.1S). Homologies of UGTs from different species focusing on regions surrounding amino acids 292, 314, 315 and 404 (Fig. 2S). Primers for UGT1A10 Mutants (Table 1S). This material is available free of charge via the Internet at http://pubs.a...

Structure and Protein–Protein Interactions of Human UDP-Glucuronosyltransferases

“…The overall three-dimensional structure of UGT1A1 showed two Rossmann fold-like domains ( Locuson and Tracy, 2007 ). Banerjee et al (2008) modeled a three-dimensional structure of human UGT1A10 using UDP-galactose 4-epimerase (1XEL) from Escherichia coli as a template structure ( Banerjee et al, 2008 ). The modeled structures of substrate- and UDP sugar-binding pockets were overlapped well with those of the template structure.…”

Revisiting the Latency of Uridine Diphosphate-Glucuronosyltransferases (UGTs)—How Does the Endoplasmic Reticulum Membrane Influence Their Function?

“…Although the complete crystal structure of a UGT protein is unsolved, homology modeling has been used to predict the three-dimensional structure [ 57 ]. For example, human UGT1A1 and UGT1A10 were homology-modeled using plant UGT71G1 and UDP-galactose 4-epimerase from Escherichia coli , respectively [ 60 , 61 ].…”

Methyl [13C]Glucopyranosiduronic Acids: Effect of COOH Ionization and Exocyclic Structure on NMR Spin-Couplings

“…Glucuronidation involves the covalent attachment of GlcUA residues to specific biomolecules in vivo to give a GlcUA conjugate, which renders toxic substances harmless and more water-soluble, and allows for their subsequent elimination via excretion . This process involves the sugar nucleotide donor, UDP- d -glucuronic acid (UDP-GlcUA), and a family of UDP-glucuronosyltransferases that catalyze the transfer of GlcUA from UDP-GlcUA to different molecular targets.…”