Crystal structure of the cofactor-binding domain of the human phase II drug-metabolism enzyme UDP-glucuronosyltransferase 2B7

Human UDP-glucuronosyltransferases (UGT) are the dominant phase II conjugative drug metabolism enzymes that also play a central role in processing 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-A 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. Designated 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 indicates human UGTs share a common catalytic mechanism. Point mutations at predicted catalytic residues in UGT2B7 abrogated activity, strongly suggesting human UGTs also utilize a serine hydrolase-like catalytic mechanism to facilitate glucuronic acid transfer.     

Identification of UDP glycosyltransferase 3A1 as a UDP N-acetylglucosaminyltransferase

The UDP glycosyltransferases (UGT) attach sugar residues to small lipophilic chemicals to alter their biological properties and enhance elimination. Of the four families present in mammals, two families, UGT1 and UGT2, use UDP glucuronic acid to glucuronidate bilirubin, steroids, bile acids, drugs, and many other endogenous chemicals and xenobiotics. UGT8, in contrast, uses UDP galactose to galactosidate ceramide, an important step in the synthesis of glycosphingolipids and cerebrosides. The function of the fourth family, UGT3, is unknown. Here we report the cloning, expression, and functional characterization of UGT3A1. This enzyme catalyzes the transfer of N-acetylglucosamine from UDP N-acetylglucosamine to ursodeoxycholic acid (3alpha, 7beta-dihydroxy-5beta-cholanoic acid). The enzyme uses ursodeoxycholic acid and UDP N-acetylglucosamine in preference to other primary and secondary bile acids, and other UDP sugars such as UDP glucose, UDP glucuronic acid, UDP galactose, and UDP xylose. In addition to ursodeoxycholic acid, UGT3A1 has activity toward 17alpha-estradiol, 17beta-estradiol, and the prototypic substrates of the UGT1 and UGT2 forms, 4-nitrophenol and 1-naphthol. A polymorphic UGT3A1 variant containing a C121G substitution was catalytically inactive. UGT3A1 is found in the liver and kidney, and to a lesser, in the gastrointestinal tract. These data describe the first characterization of a member of the UGT3 family. Its activity and distribution suggest that UGT3A1 may have an important role in the metabolism and elimination of ursodeoxycholic acid in therapies for ameliorating the symptoms of cholestasis or for dissolving gallstones.     

Identification of aspartic acid and histidine residues mediating the reaction mechanism and the substrate specificity of the human UDP-glucuronosyltransferases 1A

The human UDP-glucuronosyltransferase UGT1A6 is the primary phenol-metabolizing UDP-glucuronosyltransferase isoform. It catalyzes the nucleophilic attack of phenolic xenobiotics on UDP-glucuronic acid, leading to the formation of water-soluble glucuronides. The catalytic mechanism proposed for this reaction is an acid-base mechanism that involves an aspartic/glutamic acid and/or histidine residue. Here, we investigated the role of 14 highly conserved aspartic/glutamic acid residues over the entire sequence of human UGT1A6 by site-directed mutagenesis. We showed that except for aspartic residues Asp-150 and Asp-488, the substitution of carboxylic residues by alanine led to active mutants but with decreased enzyme activity and lower affinity for acceptor and/or donor substrate. Further analysis including mutation of the corresponding residue in other UGT1A isoforms suggests that Asp-150 plays a major catalytic role. In this report we also identified a single active site residue important for glucuronidation of phenols and carboxylic acid substrates by UGT1A enzyme family. Replacing Pro-40 of UGT1A4 by histidine expanded the glucuronidation activity of the enzyme to phenolic and carboxylic compounds, therefore, leading to UGT1A3-type isoform in terms of substrate specificity. Conversely, when His-40 residue of UGT1A3 was replaced with proline, the substrate specificity shifted toward that of UGT1A4 with loss of glucuronidation of phenolic substrates. Furthermore, mutation of His-39 residue of UGT1A1 (His-40 in UGT1A4) to proline led to loss of glucuronidation of phenols but not of estrogens. This study provides a step forward to better understand the glucuronidation mechanism and substrate recognition, which is invaluable for a better prediction of drug metabolism and toxicity in human.     

UDP-glucuronyltransferases, proteins of endoplasmic reticulum--structure and mechanism of action

UDP-glucuronyltranferase isoenzymes, UGTs, are responsible in mammals for conjugation of glucuronic acid generated by UDPGA with aglicon. UDPGA is bound to the appropriate group of I phase metabolite or, occasionally, to the native compound. As a result, the aglicon polarity increases, excreatable product in human urine is formed and, in turn, toxic effects are reduced. UGT protein structure consists of 2 domens. N-terminal catalyses of aglicon binding, whereas, C-terminal controls the addition of uridine-5'-diphosphoglucuronic acid. UGTs are anchored in endoplasmic reticulum, ER, by transmembrane fragment of C-terminal domain. The membrane location results in the latency of enzyme activity and demands specific transporters for cofactor as well as for conjugation products. There are NST and AT(ER), respectively. UDP-glucuronyltransferases exist usually as homo- and heterodimers, some of them are tetramers. 19 human UGT proteins are described by 3 gene subfamilies, UGT1A, UGT2A and UGT2B, which are expressed preferentially in the liver, but also in stomach, lung and intestine epithelium.     

Cloning and expression of human UDP-glucuronosyltransferase 1A4 in Bac-to-Bac system

UDP-glucuronosyltransferases (UGTs) catalyze the transfer of glucuronic acid from uridine diphosphate-glucuronic acid (UDP-GA) to compounds with amine, hydroxyl, and carboxylic acid moieties. N-glucuronidation is an important pathway for elimination of many tertiary amine therapeutic agents used in humans. UGT1A4 has been reported to be specific for glucuronidating primary, secondary, and tertiary amines, forming N-glucuronides. To further investigate the drugs metabolized by UGT1A4, the Bac-to-Bac expression system was used to express the recombinant UGT1A4 with His-tag on the C-terminal. The His-tagged recombinant UGT1A4 expressed in Spodoptera frugiperda (Sf9) cells were detected using anti-His antibody and the molecular weight of the recombinant protein was approximately 55kDa. The enzyme activity towards imipramine in cell homogenate protein was found to be 83.14+/-15pmol/min/mg protein (n=3) with 0.5mM imipramine by HPLC, but was not detectable in blank Sf9 cells. It paved the way for the further studies for drug glucuronidation by UGT1A4. The purification of the UGT1A4 can be done by Ni-resin. This is helpful to do research on the structure of the UFT1A4.     

The significance of amino acid residue Asp446 for enzymatic stability of rat UDP-glucuronosyltransferase UGT1A6

Asp446 in rat UDP-glucuronosyltransferase (UGT), UGT1A6, is an essential amino acid residue for its enzymatic activity (H. Iwano et al. Biochem. J. 325, 587-591, 1997). The role of Asp446 in UGT1A6 was investigated by comparing some properties of UGT mutant proteins that have a single mutation (D446K, D446E, D446N, D446Q, D446A, and D446T). These mutants, except D446K, had catalytic activities toward 1-naphthol and 4-methylumbelliferone. The UGT activities of D446E and D446N were about half of that of the wild type, and the activities of the other mutants were only about 1/5-1/10 of that of the wild type. The Km values for 1-naphthol of these mutants were similar to that of the wild type, while the values for UDP-glucuronic acid were slightly higher. The mutants were unstable in a low-pH buffer solution and were dramatically inactivated by heat treatments. Interestingly, after 30 min of treatment at 37 degrees C in the presence of UDP-glucuronic acid, the UGT activities of all functional mutants were elevated. These results suggest that Asp446 is an indispensable residue for folding a functional conformation of rat UGT1A6 by cooperation with UDP-glucuronic acid.     

Enhanced activity of <Emphasis Type="Italic">Withania somnifera</Emphasis> family-1 glycosyltransferase (UGT73A16) via mutagenesis

This work used an approach of enzyme engineering towards the improved production of baicalin as well as alteration of acceptor and donor substrate preferences in UGT73A16. The 3D model of Withania somnifera family-1 glycosyltransferase (UGT73A16) was constructed based on the known crystal structures of plant UGTs. Structural and functional properties of UGT73A16 were investigated using docking and mutagenesis. The docking studies were performed to understand the key residues involved in substrate recognition. In the molecular model of UGT73A16, substrates binding pockets are located between N- and C-terminal domains. Modeled UGT73A16 was docked with UDP-glucose, UDP-glucuronic acid (UDPGA), kaempferol, isorhamnetin, 3-hydroxy flavones, naringenin, genistein and baicalein. The protein–ligand interactions showed that His 16, Asp 246, Lys 255, Ala 337, Gln 339, Val 340, Asn 358 and Glu 362 amino acid residues may be important for catalytic activity. The kinetic parameters indicated that mutants A337C and Q339A exhibited 2–3 fold and 6–7 fold more catalytic efficiency, respectively than wild type, and shifted the sugar donor specificity from UDP-glucose to UDPGA. The mutant Q379H displayed large loss of activity with UDP-glucose and UDPGA strongly suggested that last amino acid residue of PSPG box is important for glucuronosylation and glucosylation and highly specific to sugar binding sites. The information obtained from docking and mutational studies could be beneficial in future to engineer this biocatalyst for development of better ones.     

Oligomerization of the UDP-glucuronosyltransferase 1A proteins: homo- and heterodimerization analysis by fluorescence resonance energy transfer and co-immunoprecipitation

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.     

Predicting substrate selectivity between UGT1A9 and UGT1A10 using molecular modelling and molecular dynamics approach

Uridine 5′-diphospho-glucuronosyltransferase-1A9 (UGT1A9) expressed in the liver, shows good sequence identity with UGT1A10, expressed in the intestine. Both uridine 5′-diphospho-glucuronosyltransferase (UGT) isoforms show comprehensive overlapping substrate selectivity but there are differences in stereoselectivity, regiospecificity and rate of glucuronidation of the substrates. Multiple sequence alignment analyses of UGT1A9 and UGT1A10 showed that 13% of the residues in N-terminal domain (NTD) are non-identical between them. Herein, authors attempted homology modelling of UGT1A9 and UGT1A10 and validation using software tools and reported mutagenic studies. A molecular docking study of the known substrates is performed on UGT1A9 and UGT1A10 homology models. The non-identical N-terminal residues ranging from 111 to 117 in UGT1A9 and UGT1A10 were identified to play a central role in the substrate selectivity. However, substrate binding is performed by Ser111, Gly115 and Leu117 in UGT1A10 and Gly111, Asp115 and Phe117 in UGT1A9. This study reports new residues in NTD, showing interaction with uridine 5′-diphospho-glucuronic acid which binds with C-terminal domain. Further, molecular dynamics simulations were carried out to study the role of non-identical residues in substrate identification. The study demonstrates the folding of the UGT enzyme, particularly, helix-loop-helix transition and movement of Nα3-2 helix, in response to substrate and co-substrate binding.     

Mapping the UDP-glucuronic acid binding site in UDP-glucuronosyltransferase-1A10 by homology-based modeling: confirmation with biochemical evidence

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 substrate/inhibitor, 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 the affinity ligand, 5-azidouridine-[beta- (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 the 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% less label in the 14.3 kDa peptide, respectively, compared to 1A10-His without affecting the 11.3 kDa peptide. Scatchard analysis of binding data of the affinity ligand to 1A10His-K404R and -K404E showed a 6-fold reduction and a large increase in K d, respectively. Our results indicate that 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 UDP-glcA binding to K314 and K404 in UGT1A10.     

Photoaffinity labeling of the aglycon binding site of the recombinant human liver UDP-glucuronosyltransferase UGT1A6 with 7-azido-4-methylcoumarin.

7-Azido-4-methylcoumarin (AzMC) is a fluorescent photoactive compound structurally related to 4-methylumbelliferone (4-MU), a marker substrate of the human liver recombinant UDP-glucuronosyltransferase (UGT) 1A6. AzMC was synthesized and utilized to label the substrate binding site of UGT1A6. AzMC exhibits a fluorescence spectrum with maximum excitation and emission wavelengths of 380 and 442 nm, respectively. Upon irradiation, the probe irreversibly inhibited glucuronidation activity measured with para-nitrophenol (pNP) as substrate and interacted with UGT1A6 according to a saturable process indicative of reversible binding before covalent incorporation of the photoaffinity label. This inhibition was both time and concentration dependent and led to the calculation of an inhibition constant, k(2) = 0.113 mM min(-1), and dissociation constant, K(d) = 2.89 mM, for the reaction. Partial photoinactivation of UGT1A6 with AzMC revealed that the probe decreased the apparent V(max) of the pNP glucuronidation reaction, but not the K(m). Moreover, inhibition was partially prevented by 1-naphthol, a surrogate substrate for the enzyme, or by preincubation with an active-site directed inhibitor, 5'-O-[[(2-decanoylamino-3-phenyl-propyloxycarbonyl)amino]-su lfonyl]-2 ',3'-O-isopropylideneuridine. In contrast, UDP-glucuronic acid (UDP-GlcUA) did not have any protective effect against photoinactivation and AzMC did not affect the photoaffinity labeling of UGT1A6 by 5-[beta-(32)P]N(3)UDP-GlcUA, a photoaffinity analog of UDP-GlcUA. Additionally, in the absence of irradiation, AzMC was found to be a competitive inhibitor of 4MU glucuronidation. Collectively, these results strongly indicate that AzMC specifically binds to the UGT1A6 aglycon binding site. Amino acid alignment of phenol-binding proteins revealed a conserved motif, YXXXKXXPXP. It is possible that this motif is involved in phenol binding to UGT1A6 and other phenol-accepting proteins.     

Homodimerization of human bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) and its functional implications

Genetic lesions of bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) completely or partially abolish hepatic bilirubin glucuronidation, causing Crigler-Najjar syndrome type 1 or 2, respectively. Clinical observations indicate that some mutant forms of human UGT1A1 (hUGT1A1) may be dominant-negative, suggesting their interaction with the wild-type enzyme. To evaluate intermolecular interaction of hUGT1A1, Gunn rat fibroblasts were stably transduced with hUGT1A1 cDNA. Gel permeation chromatography of solubilized microsomes suggested dimerization of hUGT1A1 in solution. Nearest-neighbor cross-linking analysis indicated that, within microsomal membranes, hUGT1A1 dimerized more efficiently at pH 7.4 than at pH 9. Two-hybrid analysis in yeast and mammalian systems demonstrated positive interaction of hUGT1A1 with itself, but not with another UGT isoform, human UGT1A6, which differs only in the N-terminal domain. Dimerization was abolished by deletion of the membrane-embedded helix from the N-terminal domain of hUGT1A1, but not by substitution of several individual amino acid residues or partial deletion of the C-terminal domain. A C127Y substitution abolished UGT1A1 activity, but not its dimerization. Coexpression of mutagenized and wild-type hUGT1A1 in COS-7 cells showed that the mutant form markedly suppressed the catalytic activity of wild-type hUGT1A1. Homodimerization of hUGT1A1 may explain the dominant-negative effect of some mutant forms of the enzyme.     

A novel glucuronyltransferase in nervous system presumably associated with the biosynthesis of HNK-1 carbohydrate epitope on glycoproteins.

Recently, embryonic chicken brain extract was shown to contain a glucuronyltransferase, which transfers glucuronic acid from UDP-glucuronic acid to glycolipid acceptors (neolactotetraosyl ceramide). The enzyme was also suggested to transfer glucuronic acid to glycoprotein acceptors (asialoorosomucoid) (Das, K. K., Basu, M., Basu, S., Chou, D. K. H., and Jungalwala, F. B. (1991) J. Biol. Chem. 266, 5238-5243). In this study, the glucuronyltransferase activity in rat brain extract was separated into two groups by UDP-glucuronic acid-Sepharose CL-6B column chromatography. The enzyme recovered predominantly in the effluent fraction (GlcAT-L) catalyzed the transfer of glucuronic acid to glycolipid acceptors but not to glycoprotein acceptors, whereas the enzyme recovered in the eluate fraction (GlcAT-P) transferred glucuronic acid most predominantly to glycoprotein acceptors and very little to glycolipid acceptors. GlcAT-P was able to transfer glucuronic acid to oligosaccharide chains on asialoorosomucoid. The enzyme recognized a terminal lactosamine structure, Gal beta 1-4GlcNAc, on glycoproteins. It was localized in the nervous system and was hardly detectable in other tissues, including the thymus, spleen, lung, kidney, and liver. Although GlcAT-L and GlcAT-P shared some properties in common such as tissue distributions and developmental changes, they exhibited marked differences in their phospholipid dependence and in their pH profiles, apart from their respective acceptor preference to glycolipids and glycoproteins. The acceptor specificity and tissue distribution suggest that a novel glucuronyltransferase, GlcAT-P, is involved in the biosynthesis of the sulfoglucuronylgalactose structure in the HNK-1 carbohydrate epitope that is expressed on glycoproteins.     

Cellular Asymmetric Catalysis by UDP-glucuronosyltransferase 1A8 Shows Functional Localization to the Basolateral Plasma Membrane

UDP-glucuronosyltransferases (UGTs) are highly expressed in liver, intestine and kidney, and catalyze the glucuronic acid conjugation of both endogenous compounds and xenobiotics. Using recombinant human UGT isoforms, we show that glucuronic acid conjugation of the model substrate, (−)-epicatechin, is catalyzed mainly by UGT1A8 and UGT1A9. In HepG2 cells, pretreatment with polyunsaturated fatty acids increased substrate glucuronidation. In the intestinal Caco-2/HT29-MTX co-culture model, overall relative glucuronidation rates were much higher than in HepG2 cells, and (−)-epicatechin was much more readily conjugated when applied to the basolateral side of the cell monolayer. Under these conditions, 95% of the conjugated product was effluxed back to the site of application, and none of the other phase 2-derived metabolites followed this distribution pattern. HT29-MTX cells contained >1000-fold higher levels of UGT1A8 mRNA than Caco-2 or HepG2 cells. Gene expression of UGT1A8 increased after treatment of cells with docosahexaenoic acid, as did UGT1A protein levels. Immunofluorescence staining and Western blotting showed the presence of UGT1A in the basal and lateral parts of the plasma membrane of HT29-MTX cells. These results suggest that some of the UGT1A8 enzyme is not residing in the endoplasmic reticulum but spans the plasma membrane, resulting in increased accessibility to compounds outside the cell. This facilitates more efficient conjugation of substrate and is additionally coupled with rapid efflux by functionally associated basolateral transporters. This novel molecular strategy allows the cell to carry out conjugation without the xenobiotic entering into the interior of the cell.     

Differential and special properties of the major human UGT1-encoded gastrointestinal UDP-glucuronosyltransferases enhance potential to control chemical uptake

UDP-glucuronosyltransferase (UGT) isozymes detoxify metabolites, drugs, toxins, and environmental chemicals via conjugation to glucuronic acid. Based on the extended UGT1 locus combined with Northern blot analysis and in situ hybridization, we determined the distribution of UGT1A1 and UGT1A7 through UGT1A10 mRNAs and found them for the first time segmentally distributed in the mucosal epithelia layer of the gastrointestinal tract. Biochemically, recombinant isozymes exhibited pH optima of 5.5, 6.4, 7.6, 8.5, and/or a broad pH range, and activities were found to be unaffected or progressively inhibited by increasing substrate concentrations after attaining Vmax for certain chemicals. Under different optimal conditions, all exhibited wide substrate selections for dietary and environmentally associated chemicals. Evidence also suggests tandem effects of isozymes in the time for completion of reactions when comparing short- and long-term incubations. Moreover, treatment of colon cells with certain diet-associated constituents, curcumin and nordihydroguaiaretic acid, reversibly targets UGTs causing inhibition without affecting protein levels; there is no direct inhibition of control UGT using curcumin as substrate in the in vitro assay. In summary, we demonstrate that UGTs are located in gastrointestinal mucosa, have vast overlapping activities under differential optimal conditions, and exhibit marked sensitivity to certain dietary substrates/constituents, representing a first comprehensive study of critical properties concerning glucuronidating isozymes in alimentary tissues. Additionally, the highly dynamic, complex, and variable properties necessarily impact absorption of ingested chemicals and therapeutic drugs.     

Cloning and characterization of a canine UDP-glucuronosyltransferase.

UDP-glucuronosyltransferases (UGTs) are a major family of enzymes catalyzing the transfer of glucuronic acid to a range of endogenous compounds and xenobiotics facilitating their elimination in either urine or bile. Although the dog is commonly used in drug metabolism studies, relatively little is known about the expression and activity of UGTs in this species. This report describes the molecular cloning and functional characterization of the first dog UGT, UGT1A6. The cloned protein is composed of 528 amino acids with the variable region demonstrating a 67-72% identity with the variable regions of mouse, rat, and human UGT1A6. The enzyme expressed stably in V79 cells predominantly catalyzed the glucuronidation of simple, planar phenols (e.g., for 1-naphthol, K(m) = 41 microM, V(max) = 0.07 nmol/min/mg protein), a class of compounds extensively glucuronidated by human UGT1A6. Based on sequence homology and common catalytic activity, this dog UGT1A protein appears to be the canine orthologue of human UGT1A6.     

Polymorphisms of the uridine-diphosphoglucuronosyltransferase 1A1 gene and coronary artery disease

Bilirubin, an antioxidant in the blood, plays a role in protection from atherosclerosis. The level of bilirubin is highly correlated to the incidence of coronary artery disease (CAD). Unconjugated bilirubin is conjugated with glucuronic acid through the reaction of uridine 5′-diphosphate-glucuronosyl transferase 1A1 (UGT1A1). The interactions of CAD and the variations in the coding regions of the UGT1A1 gene have never been evaluated. The purpose of this study was to analyze the influence of the UGT1A1 variant on the incidence of CAD. There were 135 participants in this study: 61 in the experimental group, who had CAD, and 74 in the control group, who did not have CAD. The blood samples from all 135 participants were collected and assayed to clarify the relationship between bilirubin and CAD. The assay of the polymerase chain reaction and the sequence of the UGT1A1 gene were examined to find the gene’s polymorphisms. The bilirubin levels for the participants in the control group were significantly higher than for the patients in the CAD group. Although the concentration of bilirubin in the UGT1A1 variant was higher than the wild type for the patients in the CAD group, there was no significant difference in the polymorphism of UGT1A1 between the patients in the CAD group and the participants in the control group.