Identification of the binding sites of UPD-glucuronosyltransferase 1A10.

摘要

UDP-glucuronosyltransferases (UGTs) are a family of phase II drug metabolism enzymes that transfer glucuronic acid to a variety of functional groups on the substrate, forming more soluble glucuronide that is easily excreted. UGTs accept thousands of endogenous and exogenous compounds with various structures as substrates. However, active sites for both the substrates and the common cofactor, UDP-glucuronic acid (UDP-GlcUA), are unknown. Information available on the relationship between structure and function of these enzymes is limited.; The aim of the present study is to identify amino acids localized to the substrate and co-substrate binding sites of UGT isoforms, which have been selected from a panel of recombinant His-tagged human UGT1A isoforms based on catalytic properties. To identify the phenol binding site, eight His-tagged UGT1A isoforms were analyzed in terms of their ability to glucuronidate two phenol substrates, 4-nitrophenol (pNP) and 4-methylumbelliferone (4-MU). UGT1A10 was found to be the isoform with high activity toward both phenols and, thus, was chosen for further investigation of the active sites through photoaffinity labeling followed by mass spectrometry. Several classes of photolabile probes were synthesized and evaluated as potential substrates and/or inhibitors of UGT1A isoforms to ensure the targeting to phenol active site. Based on the information obtained from those efforts, 5-azido-4-methylcoumarin (AzMC) and a benzoic acid derivative, 4-azido-2-hydroxybenzoic acid (4-AzHBA), were selected as ideal probes to photolabel UGT1A10. His-tagged UGT1A10 was solubilized and purified by Immobilized Metal Affinity Chromatography (IMAC) and then photolabeled in absence or presence of the AzMC or 4-AzHBA probes, using previously established conditions.; Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-mass spectrometry analysis of the labeled protein following digestion with trypsin identified a peptide fragment corresponding to amino acid residues 89-98 (EFMVFHAQWK) of UGT1A10 as being modified by the probes. Further analysis by ESI LC-MS/MS identified that the attachment site of the probe was localized to the quadruplet Phe90Met91-Val92-Phe93 . To confirm the involvement of the amino acids in this motif in binding with substrates, site-directed mutagenesis of Phe90 and Phe 93, two residues conserved in UGT1A7 to UGT1A10, was performed. The results of kinetic analysis of the mutants with pNP and 4-MU suggested that Phe90 and Phe93 are directly involved in the catalytic activity of UGT1A10 toward these substrates.; Analysis of UGT1A isoform activity toward estrogen and flavonoid substrates showed that UGT1A10 also had high activity toward these two classes of compounds. The phenol binding motif Phe90-Met91-Val 92-Phe93 could participate in binding of estrogens or flavonoids as well. To access this possibility, kinetic analysis of the Phe90 and Phe93 mutants toward estrogens and flavonoids were carried out. The results demonstrated common role for Phe 90-Met91-Val92-Phe93 motif in binding phenols, estrogens and flavonoids.; The UDP-GlcUA binding site is postulated to be localized in the C-terminal half of UGT molecule. Analysis of the deduced amino acid sequences of UDP-glycosyl- and glucuronosyltransferases as well as other proteins that bind sugars, nucleotides, or phosphate groups, one distinct region of homology, revealed the common motif DxxD, as a good candidate for the UDP binding site. Specifically, Asp 393 and Asp396, in UGT1A10, were determined to be the best candidates to interact with the uridinyl moiety. The roles of these two amino acids in the binding of UDP-GlcUA were investigated by site-directed mutagenesis and kinetic analysis of the mutants. The results demonstrated that mutation of Asp393 to Ala totally abolished the activity of UGT1A10 toward all substrates analyzed, suggesting a key role of this amino acid in the binding with UDP-GlcUA. Mutation of Asp396 to Ala d