4-Azido-2-hydroxybenzoic acid (4-AzHBA), a novel photoactive benzoic acid derivative, has been synthesized and used as a photoprobe to identify the phenol binding site of UDP-glucuronosyltransferases (UGTs). Analysis of recombinant His-tag UGTs from the 1A family for their ability to glucuronidate p-nitrophenol (pNP) and 4-methylumbelliferone (4-MU) revealed that UGT1A10 shows high activity toward phenols and phenol derivatives. Purified UGT1A10 was photolabeled with 4-AzHBA, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-mass spectrometry. A single modified peak corresponding to amino acid residues 89-98 (EFMVFHAQWK) of UGT1A10 was identified. The attachment site of the 4-AzHBA probe was localized to the quadruplet Phe(90)-Met(91)-Val(92)-Phe(93) using ESI LC-MS/MS. Sequence alignment revealed that the Phe(90) and Phe(93) are conserved in UGT1A7-10. Site-directed mutagenesis of these two amino acids was then followed by kinetic analysis of the mutants with two phenolic substrates, pNP and 4-MU, containing one and two planar rings, respectively. Using the combination of photoaffinity labeling, enzymatic digestion, MALDI-TOF and LC-MS mass spectrometry, and site-directed mutagenesis, we have determined for the first time that Phe(90) and Phe(93) are directly involved in the catalytic activity of UGT1A10 toward 4-MU and pNP.
Human UDP-glucuronosyltransferase 1A10 has been identified as the major isoform involved in the biotransformation of a wide range of phenolic substrates, including native estrogens and their oxidized metabolites. Our recent studies point to the F 90 -M 91 -V 92 -F 93 amino acid motif of UGT1A10, which was identified using photoaffinity labeling followed by LC-MS/MS analysis, as a key determinant of the binding of phenolic substrates. In this report, we have evaluated the role of F 90 , V 92 , and F 93 in the recognition of estrogens by UGT1A10 using site-directed mutagenesis. Kinetic studies using 5 mutants revealed that F 90 and F 93 are critical residues for the recognition of all estrogen substrates. The substitution of F 90 with alanine totally abolished the activity of this enzyme toward all the estrogens investigated. Overall, sequential removal for the aromatic (FtoL) and of the hydrophobic chain (FtoA and VtoA) from amino acids 90, 92, and 93 position effectively alters estrogen recognition. This demonstrates that individual features of the native and hydroxylated estrogens determine the specific binding properties of the compound within the binding site of the human UGT1A10 and the mutants. The resulting activities are completely abolished, unchanged, increased or decreased depending on the structures of both the mutant and the substrate. The novel identification of UGT1A10 as the major isoform involved in the glucuronidation of all estrogens and the discovery of the importance of the FMVF motif in the binding of steroids will help to elucidate the molecular mechanism of glucuronidation, resulting in the design of more effective estrogen-based therapies.
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 393
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.