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Robert H. Tukey - One of the best experts on this subject based on the ideXlab platform.
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Oligomerization of the UDP-glucuronosyltransferase 1A Proteins HOMO- AND HETERODIMERIZATION ANALYSIS BY FLUORESCENCE RESONANCE ENERGY TRANSFER AND CO-IMMUNOPRECIPITATION
The Journal of biological chemistry, 2006Co-Authors: Theresa N. Operaña, Robert H. TukeyAbstract:UDP-glucuronosyltransferases (UGTs) are membrane-bound proteins localized to the endoplasmic reticulum and catalyze the formation of beta-d-glucopyranosiduronic acids (glucuronides) using UDP-glucuronic acid and acceptor substrates such as drugs, steroids, bile acids, xenobiotics, and dietary nutrients. Recent biochemical evidence indicates that the UGT proteins may oligomerize in the membrane, but conclusive evidence is still lacking. In the present study, we have used fluorescence resonance energy transfer (FRET) to study UGT1A oligomerization in live cells. This technique demonstrated that UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 self-oligomerize (homodimerize). Heterodimer interactions were also explored, and it was determined that UGT1A1 was capable of binding with UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10. In addition to the in vivo FRET analysis, UGT1A protein-protein interactions were demonstrated through co-immunoprecipitation experiments. Co-expression of hemagglutinin-tagged and cyan fluorescent protein-tagged UGT1A proteins, followed by immunoprecipitation with anti-hemagglutinin beads, illustrated the potential of each UGT1A protein to homodimerize. Co-immunoprecipitation results also confirmed that UGT1A1 was capable of forming heterodimer complexes with all of the UGT1A proteins, corroborating the FRET results in live cells. These preliminary studies suggest that the UGT1A family of proteins form oligomerized complexes in the membrane, a property that may influence function and substrate selectivity.
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Regulation and function of family 1 and family 2 UDP-glucuronosyltransferase genes (UGT1A, UGT2B) in human oesophagus.
Biochemical Journal, 1999Co-Authors: Christian P. Strassburg, Nghia Nguyen, Michael P. Manns, Ahlke Strassburg, Robert H. TukeyAbstract:Human UDP-glucuronosyltransferases (UGTs) are expressed in a tissue-specific fashion in hepatic and extrahepatic tissues [Strassburg, Manns and Tukey (1998) J. Biol. Chem. 273, 8719-8726]. Previous work suggests that these enzymes play a protective role in chemical carcinogenesis [Strassburg, Manns and Tukey (1997) Cancer Res. 57, 2979-2985]. In this study, UGT1 and UGT2 gene expression was investigated in human oesophageal epithelium and squamous-cell carcinoma in addition to the characterization of individual UGT isoforms using recombinant protein. UGT mRNA expression was characterized by duplex reverse transcriptase-PCR analysis and revealed the expression of UGT1A7, UGT1A8, UGT1A9 and UGT1A10 mRNAs. UGT1A1, UGT1A3, UGT1A4, UGT1A5 and UGT1A6 transcripts were not detected. UGT2 expression included UGT2B7, UGT2B10 and UGT2B15, but UGT2B4 mRNA was absent. UGT2 mRNA was present at significantly lower levels than UGT1 transcripts. This observation was in agreement with the analysis of catalytic activities in oesophageal microsomal protein, which was characterized by high glucuronidation rates for phenolic xenobiotics, all of which are classical UGT1 substrates. Whereas UGT1A9 was not regulated, differential regulation of UGT1A7 and UGT1A10 mRNA was observed between normal oesophageal epithelium and squamous-cell carcinoma. Expression and analysis in vitro of recombinant UGT1A7, UGT1A9, UGT1A10, UGT2B7 and UGT2B15 demonstrated that UGT1A7, UGT1A9 and UGT1A10 catalysed the glucuronidation of 7-hydroxybenzo(alpha)pyrene, as well as other environmental carcinogens, such as 2-hydroxyamino-1-methyl-6-phenylimidazo-(4, 5-beta)-pyridine. Although UGT1A9 was not regulated in the carcinoma tissue, the five-fold reduction in 7-hydroxybenzo(alpha)pyrene glucuronidation could be attributed to regulation of UGT1A7 and UGT1A10. These data elucidate an individual regulation of human UGT1A and UGT2B genes in human oesophagus and provide evidence for specific catalytic activities of individual human UGT isoforms towards environmental carcinogens that have been implicated in cellular carcinogenesis.
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Expression of the UDP-glucuronosyltransferase 1A Locus in Human Colon: IDENTIFICATION AND CHARACTERIZATION OF THE NOVEL EXTRAHEPATIC UGT1A8 *
The Journal of biological chemistry, 1998Co-Authors: Christian P. Strassburg, Michael P. Manns, Robert H. TukeyAbstract:UDP-glucuronosyltransferases (UGT) catalyze the conjugation of lipophilic exobiotic and endobiotic compounds, which leads to the excretion of hydrophilic glucuronides via bile or urine. By a mechanism of exon sharing, the transcripts of individual first exon cassettes located at the 5′ end of the human UGT1Alocus are spliced to exons 2–5, leading to the expression of at least nine individual UGT genes. Recently, the tissue-specific expression of the UGT1A locus has been demonstrated in extrahepatic tissue, leading to the identification of UGT1A7 and UGT1A10 mRNA (Strassburg, C. P., Oldhafer, K., Manns, M. P., and Tukey, R. H. (1997) Mol. Pharmacol. 52, 212). However,UGT1A expression has not been defined in human colon, which is a metabolically active, external surface organ and a common route of drug administration. UGT1A expression was analyzed in 5 colonic, 16 hepatic, 4 biliary, and 13 gastric human tissue specimens by quantitative duplex reverse transcription-polymerase chain reaction and Western blot analysis, demonstrating lower UGT1A mRNA in the extrahepatic tissues. The precise analysis of unique UGT1A transcripts by exon 1-specific duplex reverse transcription-polymerase chain reaction revealed the expression of UGT1A1, UGT1A3, UGT1A4, UGT1A6, and UGT1A9 in the colon, which are also present in human liver. In addition, the expression of extrahepatic UGT1A10 and UGT1A8 was demonstrated. UGT1A8 was found to be closely related to gastric UGT1A7 with a 93.8% identity of first exon sequences. Expressed UGT1A7 and UGT1A10 protein showed unique catalytic activity profiles, while UGT1A8 was not active with the substrates tested. The ability of UGT1A10 to glucuronidate estrone represents only the second example of a human estrone UGT. The highly related human UGT1A7–1A10 cluster is expressed in a tissue-specific fashion and underlines the role and diversity of physiological glucuronidation at the distal end of the digestive tract.
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Polymorphic expression of the UDP-glucuronosyltransferase UGT1A gene locus in human gastric epithelium.
Molecular pharmacology, 1998Co-Authors: Christian P. Strassburg, Nghia Nguyen, Michael P. Manns, Robert H. TukeyAbstract:The human UDP-glucuronosyltransferase (UGT) 1A (UGT1A) locus is regulated in a tissue specific fashion in liver and extrahepatic tissues. Three extrahepatic UGT1A proteins, UGT1A7, UGT1A8, and UGT1A10, have been discovered and are believed to contribute to the diversity of extrahepatic glucuronidation. UGTs eliminate by glucuronidation a broad variety of endobiotic and xenobiotic substrates, which include bilirubin, therapeutic drugs, and carcinogens. Human gastric mucosa represents a primary location of tissue contact with dietary constituents, pharmaceutical drugs, and environmental carcinogens. To study the role and regulation of UGT1A gene products in stomach UGT1A mRNA expression and UGT catalytic activities were investigated in a panel of 14 normal gastric mucosa/adenocarcinoma sample pairs. UGT1A mRNA levels were differentially regulated in stomach, a feature not found in hepatic tissue. Normal gastric epithelium consistently expressed extrahepatic UGT1A7 and UGT1A10. However, polymorphic expression of UGT1A1 (29%), UGT1A3 (21%), and UGT1A6 (36%) was detected. Polymorphic UGT1A regulation was confirmed in adenocarcinoma samples with the additional observation of differential down-regulation of UGT1A1, UGT1A3, UGT1A6, and UGT1A10 and up-regulation of UGT1A7 mRNA. The polymorphic UGT1A regulation in stomach contrasts the homogeneous regulation of UGT1A gene products in human liver. Activity assays demonstrated 2- to 4-fold interindividual differences in UGT activity and qualitative differences between individuals. The polymorphic regulation of UGT1A gene products in gastric tissue may be the biological basis that determines interindividual differences in extrahepatic microsomal drug metabolism.
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Differential expression of the UGT1A locus in human liver, biliary, and gastric tissue : identification of UGT1A7 and UGT1A10 transcripts in extrahepatic tissue
Molecular pharmacology, 1997Co-Authors: Christian P. Strassburg, Michael P. Manns, Karl Oldhafer, Robert H. TukeyAbstract:Family 1 UDP-glucuronosyltransferases (UGTs) (UGT1A) are encoded by a locus that predicts the existence of at least nine individual proteins. The different proteins are generated by exon-sharing, which results in the production of a family of proteins that contain identical, 245-amino acid, carboxyl-terminal domains and an amino-terminal region of approximately 280 amino acids. The diversity of the UGT1A locus suggests the existence of complex regulation, most likely designed to account for the variable and specific glucuronidation requirements. However, the tissue-specific and extrahepatic regulation of the complete UGT1A locus has not been defined to date. In this study, quantitative duplex reverse transcription-polymerase chain reaction was used to analyze UGT1A RNA expression in 16 hepatic, four biliary, and two gastric human tissue specimens. UGT1A3 and UGT1A6 were found to be expressed in the three tissues, whereas UGT1A5 and UGT1A8 were not expressed. Hepatocellular and biliary tissue expressed UGT1A1 and UGT1A4 but hepatocellular tissue uniquely expressed UGT1A9, whereas biliary tissue expressed UGT1A10. In contrast to hepatocellular tissue, gastric tissue expressed UGT1A7 in addition to UGT1A10. The expression of UGT1A9 in hepatic tissue, UGT1A7 in gastric tissue, and UGT1A10 in biliary and gastric tissue provides evidence for the selective regulation of the UGT1A locus in hepatic and extrahepatic tissues. The newly identified UGT1A7 and UGT1A10 transcripts were cloned and found to be 95.86% identical. Sequence analysis confirmed two proteins with divergent amino termini of 285 residues and identical carboxyl termini of 245 residues. This study provides evidence for hepatic and extrahepatic regulation of the human UGT1A locus and identifies two novel extrahepatic transcripts of the UGT1A family.
Moshe Finel - One of the best experts on this subject based on the ideXlab platform.
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Molecular Docking-Based Design and Development of a Highly Selective Probe Substrate for UDP-glucuronosyltransferase 1A10.
Molecular pharmaceutics, 2018Co-Authors: Risto O. Juvonen, Moshe Finel, Johanna Troberg, Sanna Rauhamäki, Sami Kortet, Sanna Niinivehmas, Aleksanteri Petsalo, Juhani Huuskonen, Hannu Raunio, Olli T. PentikäinenAbstract:Intestinal and hepatic glucuronidation by the UDP-glucuronosyltransferases (UGTs) greatly affect the bioavailability of phenolic compounds. UGT1A10 catalyzes glucuronidation reactions in the intestine, but not in the liver. Here, our aim was to develop selective, fluorescent substrates to easily elucidate UGT1A10 function. To this end, homology models were constructed and used to design new substrates, and subsequently, six novel C3-substituted (4-fluorophenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-(dimethylamino)phenyl, 4-methylphenyl, or triazole) 7-hydroxycoumarin derivatives were synthesized from inexpensive starting materials. All tested compounds could be glucuronidated to nonfluorescent glucuronides by UGT1A10, four of them highly selectively by this enzyme. A new UGT1A10 mutant, 1A10-H210M, was prepared on the basis of the newly constructed model. Glucuronidation kinetics of the new compounds, in both wild-type and mutant UGT1A10 enzymes, revealed variable effects of the mutation. All six new C3-sub...
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UGT1A10 Is a High Activity and Important Extrahepatic Enzyme: Why Has Its Role in Intestinal Glucuronidation Been Frequently Underestimated?
Molecular pharmaceutics, 2016Co-Authors: Johanna Troberg, Ling Yang, Erkka Järvinen, Moshe FinelAbstract:The aim of this work was to highlight a considerable and broad problem in UGT1A10 activity assessment that has led to underestimation of its role in intestinal glucuronidation of drugs and other xenobiotics. The reason appears to be poor activity of the commercial UGT1A10 that is used by many laboratories, and here we have tested it by comparison with our recombinant His-tagged UGT1A10 (designated as UGT1A10-H), both expressed in insect cells. The glucuronidation rates of morphine, estradiol, estrone, SN-38, diclofenac, 4-methylumbelliferone, 7-amino-4-methylcoumarin, N-(3-carboxypropyl)-4-hydroxy-1,8-naphthalimide, and bavachinin were assayed. The results revealed that the activity of commercial UGT1A10 was low, very low, and in the cases of morphine, estrone, 7-methyl-4-aminocoumarin, and bavachinin it was below the detection limit. On the other hand, under the same conditions, UGT1A10-H exhibited high glucuronidation rates toward all these compounds. Moreover, using estradiol, morphine, and estrone, in the presence and absence of suitable inhibitors, nilotinib or atractylenolide I, it was demonstrated that UGT1A10-H, but not the commercial UGT1A10, provides a good tool to study the role of native UGT1A10 in the human intestine. The results also suggest that much of the data in the literature on UGT1A10 activity may have to be re-evaluated.
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Dog UDP-glucuronosyltransferase enzymes of subfamily 1A: cloning, expression, and activity.
Drug metabolism and disposition: the biological fate of chemicals, 2014Co-Authors: Johanna Troberg, Nina Sneitz, Erkka Järvinen, Maria Muniz, Johanna Mosorin, Marja Hagström, Moshe FinelAbstract:Understanding drug glucuronidation in the dog, a preclinical animal, is important but currently poorly characterized at the level of individual enzymes. We have constructed cDNAs for the 10 dog UDP-glucuronosyltransferases of subfamily 1A (dUGT1As), expressed them in insect cells, and assayed their activity as well as the activity of the nine human UGT1As, toward 14 compounds. The goal was to find out whether individual dUGT1As and individual human UGT1As have similar substrate specificities. The results revealed similarities but also many differences. For example, similarly to the human UGT1A10, dUGT1A11 exhibited high glucuronidation activity toward the 3-OH of 17-β-estradiol, 17-α-estradiol, and ethinylestradiol, and also conjugated the drug entacapone. Unlike the human UGT1A10, however, it failed to catalyze considerable rates of R-propranolol, diclofenac, and indomethacin glucuronidation. The estrogen glucuronidation assays revealed that dUGT1A8 and dUGT1A10 have a capacity to catalyze the formation of (linked) diglucuronides, an activity no human UGT1A exhibited. dUGT1A2-dUGT1A4 are homologs of the human UGT1A4, but none of them catalyzed N-glucuronidation of dexmedetomidine. Contrary to the human UGT1A4, however, dUGT1A2-dUGT1A4 catalyzed indomethacin and diclofenac glucuronidation. It may be concluded that, perhaps with the exception of UGT1A6, high similarities in substrate specificity between individual dog and human UGTs of subfamily 1A are rare or partial. Activity assays with liver and intestine microsomes of both dog and human further revealed interspecies differences, particularly in glucuronidation rates. In the dog, the microsomes assays also strongly suggested important roles for dUGTs of other subfamilies, mainly in the liver.
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regiospecificity and stereospecificity of human udp glucuronosyltransferases in the glucuronidation of estriol 16 epiestriol 17 epiestriol and 13 epiestradiol
Drug Metabolism and Disposition, 2013Co-Authors: Nina Sneitz, Johanna Mosorin, Mikko Vahermo, Liisa Laakkonen, Donald Poirier, Moshe FinelAbstract:The glucuronidation of estriol, 16-epiestriol, and 17-epiestriol by the human UDP-glucuronosyltransferases (UGTs) of subfamilies 1A, 2A, and 2B was examined. UGT1A10 is highly active in the conjugation of the 3-OH in all these estriols, whereas UGT2B7 is the most active UGT toward one of the ring D hydroxyls, the 16-OH in estriol and 16-epiestriol, but the 17-OH in 17-epiestriol. Kinetic analyses indicated that the 17-OH configuration plays a major role in the affinity of UGT2B7 for estrogens. The glucuronidation of the different estriols by the human liver and intestine microsomes reflects the activity of UGT1A10 and UGT2B7 in combination with the tissues’ difference in UGT1A10 expression. The UGT1A10 mutant 1A10-F93G exhibited much higher Vmax values than UGT1A10 in estriol and 17-epiestriol glucuronidation, but a significantly lower value in 16-epiestriol glucuronidation. To this study on estriol glucuronidation we have added experiments with 13-epiestradiol, a synthetic estradiol in which the spatial arrangement of the methyl on C18 and the hydroxyl on C17 is significantly different than in other estrogens. In comparison with estradiol glucuronidation, the C13 configuration change decreases the turnover of UGTs that conjugate the 3-OH, but increases it in UGTs that primarily conjugate the 17-OH. Unexpectedly, UGT2B17 exhibited similar conjugation rates of both the 17-OH and 3-OH of 13-espiestradiol. The combined results reveal the strong preference of UGT1A10 for the 3-OH of physiologic estrogens and the equivalently strong preference of UGT2B7 and UGT2B17 for the hydroxyls on ring D of such steroid hormones.
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Highly variable pH effects on the interaction of diclofenac and indomethacin with human UDP-glucuronosyltransferases.
Toxicology in vitro : an international journal published in association with BIBRA, 2012Co-Authors: Hongbo Zhang, Anne Soikkeli, Ari Tolonen, Timo Rousu, Jouni Hirvonen, Moshe FinelAbstract:Abstract In vitro glucuronidation assays of diclofenac and indomethacin at pH 7.4 are biased by the instability of the glucuronides due to acyl migration. The extent of this acyl migration may be reduced significantly by performing the glucuronidation reaction at pH 6.0. Testing the human UDP-glucuronosyltransferases (UGTs) of subfamilies 1A, 2A and 2B at pH 7.4 revealed that UGT1A10, UGT2B7 and UGT2B17 are the most active enzymes in diclofenac glucuronidation, while the highest indomethacin glucuronidation rates (corrected for relative expression levels) were exhibited by UGT2A1, UGT1A10 and UGT2B7. Interestingly, lowering the reaction pH to 6.0 increased the activity of many UGTs, particularly UGT1A10, toward both drugs, even if the rate of 4-methylumbelliferone glucuronidation by UGT1A10 at pH 6.0 was significantly lower than at pH 7.4. On the other hand, UGT2B15 lost activity upon lowering the reaction pH to 6.0. UGT1A6 does not glucuronidate diclofenac and indomethacin. Nevertheless, both drugs inhibit the 1-naphthol glucuronidation activity of UGT1A6 and their inhibition was stimulated by lowering the reaction pH, yielding significantly lower IC50 values at pH 6.0 than at pH 7.4. In conclusion, glucuronidation reactions pH affects their outcome in variable ways and could increase the toxicity of drugs that carry a carboxylic acid.
Philip Lazarus - One of the best experts on this subject based on the ideXlab platform.
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Milk Thistle Constituents Inhibit Raloxifene Intestinal Glucuronidation: A Potential Clinically Relevant Natural Product–Drug Interaction
Drug metabolism and disposition: the biological fate of chemicals, 2015Co-Authors: Brandon T. Gufford, Philip Lazarus, Gang Chen, Ana G. Vergara, Nicholas H. Oberlies, Mary F. PaineAbstract:Women at high risk of developing breast cancer are prescribed selective estrogen response modulators, including raloxifene, as chemoprevention. Patients often seek complementary and alternative treatment modalities, including herbal products, to supplement prescribed medications. Milk thistle preparations, including silibinin and silymarin, are top-selling herbal products that may be consumed by women taking raloxifene, which undergoes extensive first-pass glucuronidation in the intestine. Key constituents in milk thistle, flavonolignans, were previously shown to be potent inhibitors of intestinal UDP-glucuronosyl transferases (UGTs), with IC50s ≤ 10 μM. Taken together, milk thistle preparations may perpetrate unwanted interactions with raloxifene. The objective of this work was to evaluate the inhibitory effects of individual milk thistle constituents on the intestinal glucuronidation of raloxifene using human intestinal microsomes and human embryonic kidney cell lysates overexpressing UGT1A1, UGT1A8, and UGT1A10, isoforms highly expressed in the intestine that are critical to raloxifene clearance. The flavonolignans silybin A and silybin B were potent inhibitors of both raloxifene 4′- and 6-glucuronidation in all enzyme systems. The Kis (human intestinal microsomes, 27–66 µM; UGT1A1, 3.2–8.3 µM; UGT1A8, 19–73 µM; and UGT1A10, 65–120 µM) encompassed reported intestinal tissue concentrations (20–310 µM), prompting prediction of clinical interaction risk using a mechanistic static model. Silibinin and silymarin were predicted to increase raloxifene systemic exposure by 4- to 5-fold, indicating high interaction risk that merits further evaluation. This systematic investigation of the potential interaction between a widely used herbal product and chemopreventive agent underscores the importance of understanding natural product–drug interactions in the context of cancer prevention.
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UDP-Glucuronosyltransferase 1A10: Activity against the Tobacco-Specific Nitrosamine, 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol, and a Potential Role for a Novel UGT1A10 Promoter Deletion Polymorphism in Cancer Susceptibility
Drug metabolism and disposition: the biological fate of chemicals, 2009Co-Authors: Renee M. Balliet, Ryan W. Dellinger, Gang Chen, Philip LazarusAbstract:The extrahepatic UDP-glucuronosyltransferase 1A10 (UGT1A10) is a phase II metabolizing enzyme that is active against a number of potent carcinogens. In the present study, UGT1A10 was examined for activity against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), the major procarcinogenic metabolite of the potent tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and the promoter region of UGT1A10 was examined for variants that could lead to altered UGT1A10 expression. UGT1A10-overexpressing cell homogenates exhibited high O-glucuronidation activity against NNAL (KM = 5.95 mM). A 2000-base pair (bp) product corresponding to the UGT1A10 proximal promoter region was polymerase chain reaction (PCR)-amplified using genomic DNA from 97 white subjects, and 42 of these were sequenced. In addition to a previously reported C/G single-nucleotide polymorphism at −1271 bp (rs2741032), a novel 1664-bp deletion located between nucleotides −190 to −1856 relative to the UGT1A10 translation start site was identified. Using real-time multiplex PCR, this deletion exhibited a prevalence of 0.022 in whites (n = 156) and 0.056 in blacks (n = 133). To determine whether either polymorphism altered gene expression, in vitro assays were performed using luciferase constructs containing up to 2000 bp of the proximal UGT1A10 promoter. Constructs containing the 1664-bp deletion exhibited a significant (p = 0.009) 3-fold increase in luciferase activity compared with constructs containing the wild-type UGT1A10 promoter. No effect on luciferase activity was observed for the UGT1A10−1271G promoter variant. These data are consistent with previous studies that indicate the presence of a transcriptional repressor element within the newly identified deletion and that this deletion polymorphism may contribute to altered UGT1A10 expression and altered carcinogen detoxification between individuals.
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Glucuronidation of tobacco-specific nitrosamines by UGT2B10.
Drug Metabolism and Disposition, 2008Co-Authors: Gang Chen, Ryan W. Dellinger, Thomas E. Spratt, Philip LazarusAbstract:4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is an important tobacco-specific nitrosamine (TSNA) in the etiology of tobacco-related cancers, and N-glucuronidation is an important mechanism of NNAL detoxification. In the present study, an analysis of the UDP-glucuronosyltransferases (UGTs) responsible for the N-glucuronidation of the TSNAs N′-nitrosonornicotine, N′-nitrosoanabasine, and N′-nitrosoanatabine was performed. Using human embryonic kidney 293 cells overexpressing UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, and UGT2B17, only UGT1A4 and UGT2B10 exhibited N-glucuronidating activity against these TSNAs. The KMs for UGT2B10 were 15 to 22-fold lower than those of UGT1A4 against the three TSNAs and were similar to those observed for microsomes prepared from human liver specimens. The overall activity of UGT2B10 was 3.6 to 27-fold higher than UGT1A4 against the three TSNAs as determined by Vmax/KM after normalization by levels of UGT2B10 versus UGT1A4 mRNA. Similarly high levels of activity were also observed for UGT2B10 against a fourth TSNA, NNAL, exhibiting a 6.3-fold lower KM and 3-fold higher normalized Vmax/KM than that observed for UGT1A4. Real-time polymerase chain reaction analysis showed that UGT2B10 was expressed at a level that, on average, was 26% higher than that observed for UGT1A4 in a screening of normal liver tissue specimens from 20 individual subjects. These data suggest that UGT2B10 is likely the most active UGT isoform in human liver for the N-glucuronidation of TSNAs.
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Glucuronidation of PhIP and N-OH-PhIP by UDP-glucuronosyltransferase 1A10
Carcinogenesis, 2007Co-Authors: Ryan W. Dellinger, Gang Chen, Andrea S. Blevins-primeau, Jacek Krzeminski, Shantu Amin, Philip LazarusAbstract:The UDP-glucuronosyltransferase (UGT) 1A10 is an extra-hepatic enzyme that plays an important role in the glucuronidation of a variety of endogenous and exogenous substances and is expressed throughout the aerodigestive and digestive tracts. Two classes of carcinogens that target the colon, heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons, are known to be detoxified by the UGT family of enzymes. Recently, our laboratory demonstrated that UGT1A10 has considerably more activity against polycyclic aromatic hydrocarbons in vitro than any other UGT family member. In this study, we focused on the glucuronidation of the HCA, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and its bioactivated metabolite, N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP). We demonstrated that UGT1A10 exhibited a significantly higher glucuronidation rate against PhIP and N-OH-PhIP than any other UGT family member in vitro using whole-cell homogenates of HEK293 cells over-expressing individual UGTs. Kinetic analysis revealed a 9- and 22-fold higher level of activity for UGT1A10 homogenates as compared with the next most active UGT, UGT1A1, against N-OH-PhIP as determined by maximum rate/apparent Michaelis constant (V(max)/K(M)) at the N3 and N2 positions, respectively. The polymorphic UGT1A10(139Lys) variant exhibited a 2- to 16-fold decrease in glucuronidation activity against PhIP and N-OH-PhIP, as compared with the wild-type UGT1A10(139Glu) isoform. These data suggest that UGT1A10 is the most active UGT against PhIP and N-OH-PhIP and that UGT1A10 may play an important role in susceptibility to HCA-induced colon cancer.
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UGT1A10 promoter deletion/insertion polymorphisms: Characterization and effects of gene expression.
Cancer Epidemiology and Prevention Biomarkers, 2006Co-Authors: Renee M. Balliet, Ryan W. Dellinger, Aaron Runkle, Philip LazarusAbstract:A136 The extra-hepatic uridine-59-diphosphate-glucuronosyltransferase 1A10 (UGT1A10) is a member of the UGT1A family of genes located on 2q37 that contain common exons 2-5 with independent first exons. UGT1A10 has been previously shown to be highly active against a number of potent carcinogens including major metabolites of benzo(a)pyrene and NNK and C 18 steroids like s-estradiol. Previous experiments have described important regions of regulation of some of the UGT1A genes including UGT1A10. In the present study, the promoter region of UGT1A10 was examined for polymorphic sequence alterations that may lead to altered expression of the UGT1A10 enzyme. We fully sequenced 2,000 bp upstream of the UGT1A10 translation start site in genomic DNA specimens from 42 individual Caucasian subjects. In addition to a C/G single nucleotide polymorphism (SNP) at -1271 bp and a single thymine deletion in a thymidine repeat region located at -130 to -117 relative to the UGT1A10 translation start site that were previously identified by HapMap, a novel insertion polymorphism of two thymine nucleotides was observed at position -1538 relative to the translation start site. Polymorphic prevalences were calculated for the -1271 C>G (0.17) and the TT ins (0.07). In addition to these polymorphisms, a 1664 bp deletion located between nucleotides -191 to -1856 relative to the UGT1A10 translation start site was also identified. The presence and characterization of this deletion polymorphism was initially identified by gel electrophoresis of PCR-amplified products and characterized by direct dideoxy sequencing. This deletion had an allelic prevalence of 0.03. This deletion results in a three-fold increase in luciferase expression (p=0.01, Students t-test) as compared to the wild-type promoter in Caco-2 cells, a pattern that is consistent with previous studies demonstrating that a repressor element lies within the region spanning the newly-identified deletion. The identification of this deletion suggests that UGT1A10 may be differentially expressed between individuals and may render these subjects at altered risk for cancer. These results suggest a possible protection phenotype in individuals who contain the deletion polymorphism.
Christian P. Strassburg - One of the best experts on this subject based on the ideXlab platform.
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Regulation and function of family 1 and family 2 UDP-glucuronosyltransferase genes (UGT1A, UGT2B) in human oesophagus.
Biochemical Journal, 1999Co-Authors: Christian P. Strassburg, Nghia Nguyen, Michael P. Manns, Ahlke Strassburg, Robert H. TukeyAbstract:Human UDP-glucuronosyltransferases (UGTs) are expressed in a tissue-specific fashion in hepatic and extrahepatic tissues [Strassburg, Manns and Tukey (1998) J. Biol. Chem. 273, 8719-8726]. Previous work suggests that these enzymes play a protective role in chemical carcinogenesis [Strassburg, Manns and Tukey (1997) Cancer Res. 57, 2979-2985]. In this study, UGT1 and UGT2 gene expression was investigated in human oesophageal epithelium and squamous-cell carcinoma in addition to the characterization of individual UGT isoforms using recombinant protein. UGT mRNA expression was characterized by duplex reverse transcriptase-PCR analysis and revealed the expression of UGT1A7, UGT1A8, UGT1A9 and UGT1A10 mRNAs. UGT1A1, UGT1A3, UGT1A4, UGT1A5 and UGT1A6 transcripts were not detected. UGT2 expression included UGT2B7, UGT2B10 and UGT2B15, but UGT2B4 mRNA was absent. UGT2 mRNA was present at significantly lower levels than UGT1 transcripts. This observation was in agreement with the analysis of catalytic activities in oesophageal microsomal protein, which was characterized by high glucuronidation rates for phenolic xenobiotics, all of which are classical UGT1 substrates. Whereas UGT1A9 was not regulated, differential regulation of UGT1A7 and UGT1A10 mRNA was observed between normal oesophageal epithelium and squamous-cell carcinoma. Expression and analysis in vitro of recombinant UGT1A7, UGT1A9, UGT1A10, UGT2B7 and UGT2B15 demonstrated that UGT1A7, UGT1A9 and UGT1A10 catalysed the glucuronidation of 7-hydroxybenzo(alpha)pyrene, as well as other environmental carcinogens, such as 2-hydroxyamino-1-methyl-6-phenylimidazo-(4, 5-beta)-pyridine. Although UGT1A9 was not regulated in the carcinoma tissue, the five-fold reduction in 7-hydroxybenzo(alpha)pyrene glucuronidation could be attributed to regulation of UGT1A7 and UGT1A10. These data elucidate an individual regulation of human UGT1A and UGT2B genes in human oesophagus and provide evidence for specific catalytic activities of individual human UGT isoforms towards environmental carcinogens that have been implicated in cellular carcinogenesis.
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Expression of the UDP-glucuronosyltransferase 1A Locus in Human Colon: IDENTIFICATION AND CHARACTERIZATION OF THE NOVEL EXTRAHEPATIC UGT1A8 *
The Journal of biological chemistry, 1998Co-Authors: Christian P. Strassburg, Michael P. Manns, Robert H. TukeyAbstract:UDP-glucuronosyltransferases (UGT) catalyze the conjugation of lipophilic exobiotic and endobiotic compounds, which leads to the excretion of hydrophilic glucuronides via bile or urine. By a mechanism of exon sharing, the transcripts of individual first exon cassettes located at the 5′ end of the human UGT1Alocus are spliced to exons 2–5, leading to the expression of at least nine individual UGT genes. Recently, the tissue-specific expression of the UGT1A locus has been demonstrated in extrahepatic tissue, leading to the identification of UGT1A7 and UGT1A10 mRNA (Strassburg, C. P., Oldhafer, K., Manns, M. P., and Tukey, R. H. (1997) Mol. Pharmacol. 52, 212). However,UGT1A expression has not been defined in human colon, which is a metabolically active, external surface organ and a common route of drug administration. UGT1A expression was analyzed in 5 colonic, 16 hepatic, 4 biliary, and 13 gastric human tissue specimens by quantitative duplex reverse transcription-polymerase chain reaction and Western blot analysis, demonstrating lower UGT1A mRNA in the extrahepatic tissues. The precise analysis of unique UGT1A transcripts by exon 1-specific duplex reverse transcription-polymerase chain reaction revealed the expression of UGT1A1, UGT1A3, UGT1A4, UGT1A6, and UGT1A9 in the colon, which are also present in human liver. In addition, the expression of extrahepatic UGT1A10 and UGT1A8 was demonstrated. UGT1A8 was found to be closely related to gastric UGT1A7 with a 93.8% identity of first exon sequences. Expressed UGT1A7 and UGT1A10 protein showed unique catalytic activity profiles, while UGT1A8 was not active with the substrates tested. The ability of UGT1A10 to glucuronidate estrone represents only the second example of a human estrone UGT. The highly related human UGT1A7–1A10 cluster is expressed in a tissue-specific fashion and underlines the role and diversity of physiological glucuronidation at the distal end of the digestive tract.
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Polymorphic expression of the UDP-glucuronosyltransferase UGT1A gene locus in human gastric epithelium.
Molecular pharmacology, 1998Co-Authors: Christian P. Strassburg, Nghia Nguyen, Michael P. Manns, Robert H. TukeyAbstract:The human UDP-glucuronosyltransferase (UGT) 1A (UGT1A) locus is regulated in a tissue specific fashion in liver and extrahepatic tissues. Three extrahepatic UGT1A proteins, UGT1A7, UGT1A8, and UGT1A10, have been discovered and are believed to contribute to the diversity of extrahepatic glucuronidation. UGTs eliminate by glucuronidation a broad variety of endobiotic and xenobiotic substrates, which include bilirubin, therapeutic drugs, and carcinogens. Human gastric mucosa represents a primary location of tissue contact with dietary constituents, pharmaceutical drugs, and environmental carcinogens. To study the role and regulation of UGT1A gene products in stomach UGT1A mRNA expression and UGT catalytic activities were investigated in a panel of 14 normal gastric mucosa/adenocarcinoma sample pairs. UGT1A mRNA levels were differentially regulated in stomach, a feature not found in hepatic tissue. Normal gastric epithelium consistently expressed extrahepatic UGT1A7 and UGT1A10. However, polymorphic expression of UGT1A1 (29%), UGT1A3 (21%), and UGT1A6 (36%) was detected. Polymorphic UGT1A regulation was confirmed in adenocarcinoma samples with the additional observation of differential down-regulation of UGT1A1, UGT1A3, UGT1A6, and UGT1A10 and up-regulation of UGT1A7 mRNA. The polymorphic UGT1A regulation in stomach contrasts the homogeneous regulation of UGT1A gene products in human liver. Activity assays demonstrated 2- to 4-fold interindividual differences in UGT activity and qualitative differences between individuals. The polymorphic regulation of UGT1A gene products in gastric tissue may be the biological basis that determines interindividual differences in extrahepatic microsomal drug metabolism.
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Differential expression of the UGT1A locus in human liver, biliary, and gastric tissue : identification of UGT1A7 and UGT1A10 transcripts in extrahepatic tissue
Molecular pharmacology, 1997Co-Authors: Christian P. Strassburg, Michael P. Manns, Karl Oldhafer, Robert H. TukeyAbstract:Family 1 UDP-glucuronosyltransferases (UGTs) (UGT1A) are encoded by a locus that predicts the existence of at least nine individual proteins. The different proteins are generated by exon-sharing, which results in the production of a family of proteins that contain identical, 245-amino acid, carboxyl-terminal domains and an amino-terminal region of approximately 280 amino acids. The diversity of the UGT1A locus suggests the existence of complex regulation, most likely designed to account for the variable and specific glucuronidation requirements. However, the tissue-specific and extrahepatic regulation of the complete UGT1A locus has not been defined to date. In this study, quantitative duplex reverse transcription-polymerase chain reaction was used to analyze UGT1A RNA expression in 16 hepatic, four biliary, and two gastric human tissue specimens. UGT1A3 and UGT1A6 were found to be expressed in the three tissues, whereas UGT1A5 and UGT1A8 were not expressed. Hepatocellular and biliary tissue expressed UGT1A1 and UGT1A4 but hepatocellular tissue uniquely expressed UGT1A9, whereas biliary tissue expressed UGT1A10. In contrast to hepatocellular tissue, gastric tissue expressed UGT1A7 in addition to UGT1A10. The expression of UGT1A9 in hepatic tissue, UGT1A7 in gastric tissue, and UGT1A10 in biliary and gastric tissue provides evidence for the selective regulation of the UGT1A locus in hepatic and extrahepatic tissues. The newly identified UGT1A7 and UGT1A10 transcripts were cloned and found to be 95.86% identical. Sequence analysis confirmed two proteins with divergent amino termini of 285 residues and identical carboxyl termini of 245 residues. This study provides evidence for hepatic and extrahepatic regulation of the human UGT1A locus and identifies two novel extrahepatic transcripts of the UGT1A family.
Anna Radominska-pandya - One of the best experts on this subject based on the ideXlab platform.
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Metabolic transformation of antitumor acridinone C-1305 but not C-1311 via selective cellular expression of UGT1A10 increases cytotoxic response: implications for clinical use.
Drug Metabolism and Disposition, 2012Co-Authors: Monika Pawłowska, Anna Radominska-pandya, Barbara Fedejko-kap, Ewa Augustin, Zofia Mazerska, Timothy C. ChambersAbstract:The acridinone derivates 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone (C-1305) and 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) are promising antitumor agents with high activity against several experimental cellular and tumor models and are under evaluation in preclinical and early phase clinical trials. Recent evidence from our laboratories has indicated that both compounds were conjugated by several uridine diphosphate-glucuronyltransferase (UGT) isoforms, the most active being extrahepatic UGT1A10. The present studies were designed to test the ability and selectivity of UGT1A10 in the glucuronidation of acridinone antitumor agents in a cellular context. We show that in KB-3 cells, a HeLa subline lacking expression of any UGT isoforms, both C-1305 and C-1311 undergo metabolic transformation to the glucuronidated forms on overexpression of UGT1A10. Furthermore, UGT1A10 overexpression significantly increased the cytotoxicity of C-1305, but not C-1311, suggesting that the glucuronide was more potent than the C-1305 parent compound. These responses were selective for UGT1A10 because documented overexpression of UGT2B4 failed to produce glucuronide products and failed to alter the cytotoxicity for both compounds. These findings contribute to our understanding of the mechanisms of action of these agents and are of particular significance because data for C-1305 contradict the dogma that glucuronidation typically plays a role in detoxification or deactivation. In summary, these studies suggest that extrahepatic UGT1A10 plays an important role in the metabolism and the bioactivation of C-1305 and constitutes the basis for further mechanistic studies on the mode of action of this drug, as well as translational studies on the role of this enzyme in regulation of C-1305 toxicity in cancer.
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Phenylalanine 93 of the human UGT1A10 plays a major role in the interactions of the enzyme with estrogens.
Steroids, 2011Co-Authors: Camilla Höglund, Anna Radominska-pandya, Nina Sneitz, Liisa Laakonen, Moshe FinelAbstract:Little is currently known about the substrate binding site of the human UDP-glucuronosyltransferases (UGTs) and the structural elements that affect their complex substrate selectivity. In order to further understand and extend our earlier findings with phenylalanines 90 and 93 of UGT1A10, we have replaced each of them with Gly, Ala, Val, Leu, Ile or Tyr, and tested the activity of the resulting 12 mutants toward eight different substrates. Apart from scopoletin glucuronidation, the F90 mutants other than F90L were nearly inactive, while the F93 mutants’ activity was strongly substrate dependent. Hence, F93L displayed high entacapone and 1-naphthol glucuronidation rates, whereas F93G, which was nearly inactive in entacapone glucuronidation, was highly active toward estradiol, estriol and even ethinylestradiol, a synthetic estrogen that is a poor substrate for the wild-type UGT1A10. Kinetic analyses of 4-nitrophenol, estradiol and ethinylestradiol glucuronidation by the mutants that catalyzed the respective reactions at considerable rates, revealed increased Km values for 4-nitrophenol and estradiol in all the mutants, whilst the Km values of F93G and F93A for ethinylestradiol were lower than in control UGT1A10. Based on the activity results and a new molecular model of UGT1A10, it is suggested that both F90 and F93 are located in a surface helix at the far end of the substrate binding site. Nevertheless, only F93 directly affects the selectivity of UGT1A10 toward large and rigid estrogens, particularly those with substitutions at the D ring. The effects of F93 mutations on the glucuronidation of smaller or less rigid substrates are indirect, however.
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Characterization of human hepatic and extrahepatic UDP-glucuronosyltransferase enzymes involved in the metabolism of classic cannabinoids.
Drug metabolism and disposition: the biological fate of chemicals, 2009Co-Authors: Anna Mazur, Moshe Finel, Cheryl F. Lichti, Paul L. Prather, Agnieszka K. Zielinska, Stacie M. Bratton, Anna Gallus-zawada, Grover P. Miller, Anna Radominska-pandya, Jeffery H. MoranAbstract:Tetrahydrocannabinol (Δ9-THC), the primary psychoactive ingredient in marijuana, is subject to cytochrome P450 oxidation and subsequent UDP-glucuronosyltransferase (UGT)-dependent glucuronidation. Many studies have shown that CYP2C9 and CYP3A4 are the primary enzymes responsible for these cytochrome P450-dependent oxidations, but little work has been done to characterize phase II metabolic pathways. In this study, we test the hypothesis that there are specific human UGTs responsible for classic cannabinoid metabolism. The activities of 12 human recombinant UGTs toward classic cannabinoids [cannabinol (CBN), cannabidiol (CBD), (–)-Δ8-THC, (–)-Δ9-THC, (±)-11-hydroxy-Δ9-THC (THC-OH), and (–)-11-nor-9-carboxy-Δ9-THC (THC-COOH)] were evaluated using high-performance liquid chromatography-tandem mass spectrometry and labeling assays. Despite activity by UGT1A1, 1A3, 1A8, 1A9, 1A10, and 2B7 toward CBN, CBD, THC-OH, and THC-COOH, only selected UGTs demonstrate sufficient activity for further characterization of steady-state kinetics. CBN was the most recognized substrate as evidenced by activities from hepatic UGT1A9 and extrahepatic UGT1A7, UGT1A8, and UGT1A10. These results may reflect the introduction of an aromatic ring to Δ9-THC, leading to favorable π stacking with phenylalanines in the UGT active site. Likewise, oxidation of Δ9-THC to THC-OH results in UGT1A9 and UGT1A10 activity toward the cannabinoid. Further oxidation to THC-COOH surprisingly leads to a loss in metabolism by UGT1A9 and UGT1A10, while creating a substrate recognized by UGT1A1 and UGT1A3. The resulting glucuronide of THC-COOH is the main metabolite found in urine, and thus these hepatic enzymes play a critical role in the metabolic clearance of cannabinoids. Taken together, glucuronidation of cannabinoids depends on upstream processing including enzymes such as CYP2C9 and CYP3A4.
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Identification of UDP-glucuronosyltransferase 1A10 in non-malignant and malignant human breast tissues.
Steroids, 2008Co-Authors: Athena Starlard-davenport, Beverly Lyn-cook, Anna Radominska-pandyaAbstract:Abstract UGT1A10 was recently identified as the major isoform that conjugates estrogens. In this study, real-time PCR revealed high levels of UGT1A10 and UGT2B7 mRNA in human breast tissues. The expression of UGT1A10 in breast was a novel finding. UGT1A10 and UGT2B7 mRNAs were differentially expressed among normal and malignant specimens. Their overall expression was significantly decreased in breast carcinomas as compared to normal breast specimens (UGT1A10: 68 ± 26 vs. 252 ± 86, respectively; p
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The first aspartic acid of the DQxD motif for human UDP-glucuronosyltransferase 1A10 interacts with UDP-glucuronic acid during catalysis.
Drug metabolism and disposition: the biological fate of chemicals, 2007Co-Authors: Yan Xiong, Moshe Finel, Agnieszka K. Zielinska, Stacie M. Bratton, Grover P. Miller, Anne-sisko Patana, Michael J. Miley, Adrian Goldman, Matthew R. Redinbo, Anna Radominska-pandyaAbstract: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 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. Although D393 (D398 in UGT2B7) is similarly critical for UDP-GlcUA binding in both enzymes, the effects of Q394 (Q399 in UGT2B7) to Ala mutation on activity were significant but different between UGT1A10 and UGT2B7. A model of the UDP-GlcUA binding site suggests that the contribution of other residues to cosubstrate 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 cosubstrate 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.
