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This study delves into the intricate mechanisms underlying drug-induced liver injury (DILI) with a specific focus on bromfenac, the withdrawn nonsteroidal anti-inflammatory drug. DILI is a pervasive concern in drug development, prompting market withdrawals and posing significant challenges to healthcare. Despite the withdrawal of bromfenac due to DILI, the exact role of its microsomal metabolism in inducing hepatotoxicity remains unclear. Herein, employing HepG2 cells with human liver microsomes and UDP-glucuronic acid (UDPGA), our investigation revealed a substantial increase in bromfenac-induced cytotoxicity in the presence of UDPGA, pointing to the significance of UDP-glucuronosyltransferase (UGT)-dependent metabolism in augmenting toxicity. Notably, among the recombinant UGTs examined, UGT2B7 emerged as a pivotal enzyme in the metabolic activation of bromfenac. Metabolite identification studies disclosed the formation of reactive intermediates, with bromfenac indolinone (lactam) identified as a potential mediator of hepatotoxic effects. Moreover, in cytotoxicity experiments, the toxicity of bromfenac lactam exhibited a 34-fold increase, relative to bromfenac. The toxicity of bromfenac lactam was mitigated by nicotinamide adenine dinucleotide phosphate-dependent metabolism. This finding underscores the role of UGT-dependent metabolism in generating reactive metabolites that contribute to the observed hepatotoxicity associated with bromfenac. Understanding these metabolic pathways and the involvement of specific enzymes, such as UGT2B7, provides crucial insights into the mechanisms of bromfenac-induced liver injury. In conclusion, this research sheds light on the metabolic intricacies leading to cytotoxicity induced by bromfenac, especially emphasizing the role of UGT-dependent metabolism and the formation of reactive intermediates like bromfenac lactam. These findings offer insight into the mechanistic basis of DILI and emphasize the importance of understanding metabolism-mediated toxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

UXS1-mediated clearance of the sugar nucleotide UDPGA is a specific vulnerability of cancer cells., (©2023 American Association for Cancer Research.)

Uridine diphosphate glucuronic acid (UDPGA) is an essential substrate in the glucuronidation of exogenous and endogenous lipophilic compounds via the liver glucuronic acid pathway, and its synthesis depends on glucose and energy in the body. Bisphenol S (BPS), as a lipophilic environmental pollutant, has been widely utilized in the manufacturing of daily necessities. The biological effect of BPS in interference with liver energy metabolism might affect UDPGA synthesis and the excretion of lipophilic compounds, but this was not clearly revealed. Here, female zebrafish that were exposed to BPS for 35 days exhibited a significant decrease in UDPGA in the liver with significant accumulation of exogenous BPS and endogenous bilirubin in the body. One vital reason may be that the exposure to BPS for 35 days promoted the lipid formation through PPARg signaling and reduced energy levels in the liver, resulting in the decreased raw materials for UDPGA production in glucuronic acid pathway. Meanwhile, transcriptome analysis showed that BPS inhibited the mRNA expression levels of genes related to the glucuronic acid pathway. The accumulation of endogenous and exogenous lipophilic compounds can trigger a variety of toxicological effect. Thus, weakened liver detoxification might be the primary cause of the toxicological effects of lipophilic pollutants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Identifying metabolic steps that are specifically required for the survival of cancer cells but are dispensable in normal cells remains a challenge 1 . Here we report a therapeutic vulnerability in a sugar nucleotide biosynthetic pathway that can be exploited in cancer cells with only a limited impact on normal cells. A systematic examination of conditionally essential metabolic enzymes revealed that UXS1, a Golgi enzyme that converts one sugar nucleotide (UDP-glucuronic acid, UDPGA) to another (UDP-xylose), is essential only in cells that express high levels of the enzyme immediately upstream of it, UGDH. This conditional relationship exists because UXS1 is required to prevent excess accumulation of UDPGA, which is produced by UGDH. UXS1 not only clears away UDPGA but also limits its production through negative feedback on UGDH. Excess UDPGA disrupts Golgi morphology and function, which impedes the trafficking of surface receptors such as EGFR to the plasma membrane and diminishes the signalling capacity of cells. UGDH expression is elevated in several cancers, including lung adenocarcinoma, and is further enhanced during chemoresistant selection. As a result, these cancer cells are selectively dependent on UXS1 for UDPGA detoxification, revealing a potential weakness in tumours with high levels of UGDH., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Polymyxins are important last resort antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. However, pathogens have acquired resistance to polymyxins through a pathway that modifies lipid A with 4-amino-4-deoxy-l-arabinose (Ara4N). Inhibition of this pathway is, therefore, a desirable strategy to combat polymyxin resistance. The first pathway-specific reaction is an NAD + -dependent oxidative decarboxylation of UDP-glucuronic acid (UDP-GlcA) catalyzed by the dehydrogenase domain of ArnA (ArnA_DH). We present the crystal structure of Salmonella enterica serovar typhimurium ArnA in complex with UDP-GlcA showing that binding of the sugar nucleotide is sufficient to trigger a conformational change conserved in bacterial ArnA_DHs but absent in its human homologs, as confirmed by structure and sequence analysis. Ligand binding assays show that the conformational change is essential for NAD + binding and catalysis. Enzyme activity and binding assays show that (i) UDP-GlcA analogs lacking the 6' carboxylic acid bind the enzyme but fail to trigger the conformational change, resulting in poor inhibition, and (ii) the uridine monophosphate moiety of the substrate provides most of the ligand binding energy. Mutation of asparagine 492 to alanine (N492A) disrupts the ability of ArnA_DH to undergo the conformational change while retaining substrate binding, suggesting that N492 is involved in sensing the 6' carboxylate in the substrate. These results identify the UDP-GlcA-induced conformational change in ArnA_DH as an essential mechanistic step in bacterial enzymes, providing a platform for selective inhibition.

Uridine diphosphate glucose (UDP-Glc) is able to accelerate the decay of snail family transcriptional repressor 1 ( SNAI1 ) mRNA by inhibiting Hu antigen R (HuR, an RNA-binding protein), thereby preventing cancer invasiveness and drug resistance. Nevertheless, the phosphorylation of tyrosine 473 (Y473) of UDP-glucose dehydrogenase (UGDH is capable of converting UDP-Glc to uridine diphosphate glucuronic acid (UDP-GlcUA)) weakens the inhibition of UDP-Glc to HuR, thus initiating the epithelial-mesenchymal transformation of tumor cells and promoting tumor cell migration and metastasis. To address the mechanism, we performed molecular dynamics simulations combined with molecular mechanics generalized Born surface area (MM/GBSA) analysis on wild-type and Y473 phosphorylated UGDH and HuR, UDP-Glc, UDP-GlcUA complexes. We demonstrated that Y473 phosphorylation was able to enhance the binding between UGDH and the HuR/UDP-Glc complex. Compared with HuR, UGDH has a stronger binding ability with UDP-Glc; therefore, UDP-Glc was inclined to bind to UGDH and then was catalyzed to UDP-GlcUA by UGDH, which relieved the inhibition of UDP-Glc to HuR. In addition, the binding ability of HuR for UDP-GlcUA was lower than its affinity for UDP-Glc, significantly reducing the inhibition of HuR. Hence, HuR bound to SNAI1 mRNA more easily to increase the stability of mRNA. Our results revealed the micromolecular mechanism of Y473 phosphorylation of UGDH regulating the interaction between UGDH and HuR as well as relieving the inhibition of UDP-Glc on HuR, which contributed to understanding the role of UGDH and HuR in tumor metastasis and developing small molecule drugs targeting the interaction between UGDH and HuR.

UDP-glucuronic acid (UDP-GlcA) 4-epimerase illustrates an important problem regarding enzyme catalysis: balancing conformational flexibility with precise positioning. The enzyme coordinates the C4-oxidation of the substrate by NAD + and rotation of a decarboxylation-prone β-keto acid intermediate in the active site, enabling stereoinverting reduction of the keto group by NADH. We reveal the elusive rotational landscape of the 4-keto intermediate. Distortion of the sugar ring into boat conformations induces torsional mobility in the enzyme's binding pocket. The rotational endpoints show that the 4-keto sugar has an undistorted 4 C 1 chair conformation. The equatorially placed carboxylate group disfavors decarboxylation of the 4-keto sugar. Epimerase variants lead to decarboxylation upon removal of the binding interactions with the carboxylate group in the opposite rotational isomer of the substrate. Substitutions R185A/D convert the epimerase into UDP-xylose synthases that decarboxylate UDP-GlcA in stereospecific, configuration-retaining reactions., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Background: Saccharolactone is used as a β-glucuronidase inhibitor in in vitro microsomal and recombinant uridine diphosphoglucuronosyl transferases (rUGTs) incubations to enhance glucuronide pathway and, thereby, formation of glucuronide metabolites. We investigated its effect on CYP mediated metabolism of drugs (compound-174, phenacetin and quinidine) using human liver microsomes (HLM) supplemented with Phase-1 and Phase-2 co-factors., Methods: Compounds were incubated in HLM supplemented with co-factors to assess Phase-1 (NADPH) and Phase-2 (NADPH, alamethicin, saccharolactone and UDPGA) metabolism. CYP phenotype assay for compound-174 was conducted in HLM (± 1-ABT) and human recombinant CYP isoforms. CYP inhibition profile of saccharolactone was also generated in HLM., Results: The metabolism of compound-174, phenacetin and quinidine in HLM significantly decreased in reactions containing additional components like alamethicin, saccharolactone and UDPGA and indicated that the addition of saccharolactone inhibited the metabolism. Phenacetin and quinidine are known substrates of CYP1A2 and CYP3A4 isoforms. The metabolism of compound- 174 was significantly inhibited in the presence of 1-ABT in HLM, and CYP3A4 and CYP2C8 isoforms were found to be the predominant isoforms responsible for its metabolism. Further evaluation of CYP inhibition in HLM indicated saccharolactone to be a strong inhibitor of CYP1A2, 2D6, 3A4 and 2C8 isoforms with IC 50 values of less than 4 mM., Conclusion: The findings indicated that saccharolactone being a strong inhibitor of CYP1A2, 2D6, 3A4 and 2C8 isoforms (IC 50 < 4 mM), resulted in significant inhibition of the metabolism of compound-174, phenacetin and quinidine in HLM and caution should be exercised in using it with proper titration of the concentrations., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Uridine diphosphate glucuronosyltransferase (UGT) enzymes conjugate many lipophilic chemicals, such as drugs, environmental contaminants, and endogenous compounds, promoting their excretion. The complexity of UGT kinetics, and the location of enzyme active site in endoplasmic reticulum lumen, requires an accurate optimisation of enzyme assays.In the present study, we characterised UGT activity in liver microsomes of green turtles ( Chelonia mydas ), an endangered species. The conditions for measuring UGT activity were standardised through spectrofluorimetric methods, using the substrates 4-methylumbelliferone (4-MU) and uridine diphosphate glucuronic acid (UDPGA) at 30 °C and pH 7.4.The green turtles showed UGT activity at the saturating concentrations of substrates of 250 µM to 4-MU and 7 mM to UDPGA. The alamethicin, Brij ® 58, bovine serum albumin (BSA), and magnesium increased UGT activity. The assay using alamethicin (22 µg per mg of protein), magnesium (1 mM), and BSA (0.25%) reached the highest V max (1203 pmol·min -1 mg·protein -1 ). Lithocholic acid and diclofenac inhibited UGT activity in green turtles.This study is the first report of UGT activity in the liver of green turtles and provides a base for future studies to understand the mechanisms of toxicity by exposure to contaminants in this charismatic species.

The antifungal agent 5-fluorocytosine (5-FC) is used for the treatment of several mycoses, but is unsuitable for monotherapy due to the rapid development of resistance. Here, we show that cryptococci develop resistance to 5-FC at a high frequency when exposed to concentrations several fold above the minimal inhibitory concentration. The genomes of resistant clones contain alterations in genes relevant as well as irrelevant for 5-FC resistance, suggesting that 5-FC may be mutagenic at moderate concentrations. Mutations in FCY2 (encoding a known permease for 5-FC uptake), FCY1, FUR1, UXS1 (encoding an enzyme that converts UDP-glucuronic acid to UDP-xylose) and URA6 contribute to 5-FC resistance. The uxs1 mutants accumulate UDP-glucuronic acid, which appears to down-regulate expression of permease FCY2 and reduce cellular uptake of the drug. Additional mutations in genes known to be required for UDP-glucuronic acid synthesis (UGD1) or a transcriptional factor NRG1 suppress UDP-glucuronic acid accumulation and 5-FC resistance in the uxs1 mutants.

Hyaluronan (HA) is a linear glycosaminoglycan (GAG) of extracellular matrix (ECM) synthesized by three hyaluronan synthases (HASes) at the plasma membrane using uridine diphosphate (UDP)-glucuronic acid (UDP-GlcUA) and UDP- N- acetylglucosamine (UDP-GlcNAc) as substrates. The production of HA is mainly regulated by hyaluronan synthase 2 (HAS2), that can be controlled at different levels, from epigenetics to transcriptional and post-translational modifications. HA biosynthesis is an energy-consuming process and, along with HA catabolism, is strongly connected to the maintenance of metabolic homeostasis. The cytoplasmic pool of UDP-sugars is critical for HA synthesis. UDP-GlcNAc is an important nutrient sensor and serves as donor substrate for the O -GlcNAcylation of many cytosolic proteins, including HAS2. This post-translational modification stabilizes HAS2 in the membrane and increases HA production. Conversely, HAS2 can be phosphorylated by AMP activated protein kinase (AMPK), a master metabolic regulator activated by low ATP/AMP ratios, which inhibits HA secretion. Similarly, HAS2 expression and the deposition of HA within the pericellular coat are inhibited by sirtuin 1 (SIRT1), another important energetic sensor, confirming the tight connection between nutrients availability and HA metabolism.

Glucuronidation, catalyzed by uridine diphosphate glucuronosyltransferases (UGTs), is an important process for the metabolism and clearance of many lipophilic chemicals, including drugs, environmental chemicals, and endogenous compounds. Glucuronidation is a bisubstrate reaction that requires the aglycone and the cofactor, UDP-GlcUA. Accumulating evidence suggests that the bisubstrate reaction follows a compulsory-order ternary mechanism. To simplify the kinetic modeling of glucuronidation reactions in vitro, UDP-GlcUA is usually added to incubations in large excess. Many factors have been shown to influence UGT activity and kinetics in vitro, and these must be accounted for during experimental design and data interpretation. While the assessment of drug-drug interactions resulting from UGT inhibition has been challenging in the past, the increasing availability of UGT enzyme-selective substrate and inhibitor "probes" provides the prospect for more reliable reaction phenotyping and assessment of drug-drug interaction potential. Although extrapolation of the in vitro intrinsic clearance of a glucuronidated drug often underpredicts in vivo clearance, careful selection of in vitro experimental conditions and inclusion of extrahepatic glucuronidation may improve the predictivity of in vitro-in vivo extrapolation. Physiologically based pharmacokinetic (PBPK) modeling has also shown to be of value for predicting PK of drugs eliminated by glucuronidation., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Enzymes of the human UDP-glycosyltransferase (UGT) superfamily typically catalyze the covalent addition of the sugar moiety from a UDP-sugar cofactor to relatively low-molecular weight lipophilic compounds. Although UDP-glucuronic acid (UDP-GlcUA) is most commonly employed as the cofactor by UGT1 and UGT2 family enzymes, UGT2B7 and several other enzymes can use both UDP-GlcUA and UDP-glucose (UDP-Glc), leading to the formation of glucuronide and glucoside conjugates. An investigation of UGT2B7-catalyzed morphine glycosidation indicated that glucuronidation is the principal route of metabolism because the binding affinity of UDP-GlcUA is higher than that of UDP-Glc. Currently, it is unclear which residues in the UGT2B7 cofactor binding domain are responsible for the preferential binding of UDP-GlcUA. Here, molecular dynamics (MD) simulations were performed together with site-directed mutagenesis and enzyme kinetic studies to identify residues within the UGT2B7 binding site responsible for the selective cofactor binding. MD simulations demonstrated that Arg259, which is located within the N-terminal domain, specifically interacts with UDP-GlcUA, whereby the side chain of Arg259 H-bonds and forms a salt bridge with the carboxylate group of glucuronic acid. Consistent with the MD simulations, substitution of Arg259 with Leu resulted in the loss of morphine, 4-methylumbelliferone, and zidovudine glucuronidation activity, but morphine glucosidation was preserved. SIGNIFICANCE STATEMENT: Despite the importance of uridine diphosphate glycosyltransferase (UGT) enzymes in drug and chemical metabolism, cofactor binding interactions are incompletely understood, as is the molecular basis for preferential glucuronidation by UGT1 and UGT2 family enzymes. The study demonstrated that long timescale molecular dynamics (MD) simulations with a UGT2B7 homology model can be used to identify critical binding interactions of a UGT protein with UDP-sugar cofactors. Further, the data provide a basis for the application of MD simulations to the elucidation of UGT-aglycone interactions., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Triterpenoid saponins are specialised metabolites distributed widely in the plant kingdom that consist of one or more sugar moieties attached to triterpenoid aglycones. Despite the widely accepted view that glycosylation is catalysed by UDP-dependent glycosyltransferase (UGT), the UGT which catalyses the transfer of the conserved glucuronic acid moiety at the C-3 position of glycyrrhizin and various soyasaponins has not been determined. Here, we report that a cellulose synthase superfamily-derived glycosyltransferase (CSyGT) catalyses 3-O-glucuronosylation of triterpenoid aglycones. Gene co-expression analyses of three legume species (Glycyrrhiza uralensis, Glycine max, and Lotus japonicus) reveal the involvement of CSyGTs in saponin biosynthesis, and we characterise CSyGTs in vivo using Saccharomyces cerevisiae. CSyGT mutants of L. japonicus do not accumulate soyasaponin, but the ectopic expression of endoplasmic reticulum membrane-localised CSyGTs in a L. japonicus mutant background successfully complement soyasaponin biosynthesis. Finally, we produced glycyrrhizin de novo in yeast, paving the way for sustainable production of high-value saponins.

The transport of UDP-glucuronic acid (UDPGA), a co-substrate of UDP-glucuronosyltransferase (UGT), to the intraluminal side of the endoplasmic reticulum (ER) is an essential step in the glucuronidation of exogenous and endogenous compounds. According to a previous study, the expression of recombinant SLC35B1, SLC35B4, or SLC35D1, nucleotide sugar transporters, in V79 cells has the potential to transport UDPGA into the lumen of microsomes. The purpose of this study is to examine whether the transport of UDPGA by these transporters substantially affects UGT activity. Since the knockdown of UDP-glucose 6-dehydrogenase, a synthetase of UDPGA, in HEK293 cells stably expressing UGT1A1 (HEK/UGT1A1 cells) resulted in a significant decrease in 4-methylumbelliferone (4-MU) glucuronosyltransferase activity, supplementation of a sufficient amount of UDPGA is required for UGT activity. By performing qRT-PCR using cDNA samples from 21 human liver samples, we observed levels of the SLC35B1 and SLC35D1 mRNAs that were 15- and 14-fold higher, respectively, than the levels of the SLC35B4 mRNA, and SLC35B1 showed the largest (37-fold) interindividual variability. Interestingly, 4-MU glucuronosyltransferase activity was significantly decreased upon the knockdown of SLC35B1 in HEK/UGT1A1 cells, and this phenomenon was also observed in HepaRG cells. Using siRNAs targeting 23 different SLC35 subfamilies, the knockdown of SLC35B1 and SLC35E3 decreased 4-MU glucuronosyltransferase activity in HEK/UGT1A1 cells. However, the 4-MU glucuronosyltransferase activity was not altered by SLC35E3 knockdown in HepaRG cells, suggesting that SLC35B1 was the main transporter of UDPGA into the ER in the human liver. In conclusion, SLC35B1 is a key modulator of UGT activity by transporting UDPGA to the intraluminal side of the ER., (Copyright © 2020 Elsevier Inc. All rights reserved.)

An improved understanding of the biochemical alterations that accompany tumor progression and metastasis is necessary to inform the next generation of diagnostic tools and targeted therapies. Metabolic reprogramming is known to occur during the epithelial-mesenchymal transition (EMT), a process that promotes metastasis. Here, we identify metabolic enzymes involved in extracellular matrix remodeling that are upregulated during EMT and are highly expressed in patients with aggressive mesenchymal-like breast cancer. Activation of EMT significantly increases production of hyaluronic acid, which is enabled by the reprogramming of glucose metabolism. Using genetic and pharmacological approaches, we show that depletion of the hyaluronic acid precursor UDP-glucuronic acid is sufficient to inhibit several mesenchymal-like properties including cellular invasion and colony formation in vitro, as well as tumor growth and metastasis in vivo. We found that depletion of UDP-glucuronic acid altered the expression of PPAR-gamma target genes and increased PPAR-gamma DNA-binding activity. Taken together, our findings indicate that the disruption of EMT-induced metabolic reprogramming affects hyaluronic acid production, as well as associated extracellular matrix remodeling and represents pharmacologically actionable target for the inhibition of aggressive mesenchymal-like breast cancer progression.

Liver X receptors (LXRs), LXR α and LXR β , are nuclear receptors that regulate the metabolism of cholesterol and bile acids and are activated by oxysterols. Humanized UGT1 ( hUGT1 ) mice express the 9-human UGT1A genes associated with the UGT1 locus in a Ugt1 -null background. The expression of UGT1A1 is developmentally delayed in the liver and intestines, resulting in the accumulation of serum bilirubin during the neonatal period. Induction of UGT1A1 in newborn hUGT1 mice leads to rapid reduction in total serum bilirubin (TSB) levels, a phenotype measurement that allows for an accurate prediction on UGT1A1 expression. When neonatal hUGT1 mice were treated by oral gavage with the LXR agonist T0901317, TSB levels were dramatically reduced. To determine the LXR contribution to the induction of UGT1A1 and the lowering of TSB levels, experiments were conducted in neonatal hUGT1/Lxrα -/- , hUGT1/Lxrβ -/- , and hUGT1/Lxrαβ -/- mice treated with T0901317. Induction of liver UGT1A1 was dependent upon LXR α , with the induction pattern paralleling induction of LXR α -specific stearoyl CoA desaturase 1. However, the actions of T0901317 were also shown to display a lack of specificity for LXR, with the induction of liver UGT1A1 in hUGT1/Lxrαβ -/- mice, a result associated with activation of both pregnane X receptor and constitutive androstane receptor. However, the LXR agonist GW3965 was highly selective toward LXR α , showing no impact on lowering TSB values or inducing UGT1A1 in hUGT1/Lxrα -/- mice. An LXR-specific enhancer site on the UGT1A1 gene was identified, along with convincing evidence that LXR α is crucial in maintaining constitutive expression of UGT1A1 in adult hUGT1 mice. SIGNIFICANCE STATEMENT: It has been established that activation of LXR α , and not LXR β , is responsible for the induction of liver UGT1A1 and metabolism of serum bilirubin in neonatal hUGT1 mice. Although induction of the human UGT1A1 gene is initiated at a newly characterized LXR enhancer site, allelic deletion of the Lxrα gene drastically reduces the constitutive expression of liver UGT1A1 in adult hUGT1 mice. Combined, these findings indicate that LXR α is critical for the developmental expression of UGT1A1., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

A highly efficient di- C -glycosyltransferase GgCGT was discovered from the medicinal plant Glycyrrhiza glabra . GgCGT catalyzes a two-step di- C -glycosylation of flopropione-containing substrates with conversion rates of >98%. To elucidate the catalytic mechanisms of GgCGT, we solved its crystal structures in complex with UDP-Glc, UDP-Gal, UDP/phloretin, and UDP/nothofagin, respectively. Structural analysis revealed that the sugar donor selectivity was controlled by the hydrogen-bond interactions of sugar hydroxyl groups with D390 and other key residues. The di- C -glycosylation capability of GgCGT was attributed to a spacious substrate-binding tunnel, and the G389K mutation could switch di- to mono- C -glycosylation. GgCGT is the first di- C -glycosyltransferase with a crystal structure, and the first C -glycosyltransferase with a complex structure containing a sugar acceptor. This work could benefit the development of efficient biocatalysts to synthesize C -glycosides with medicinal potential.

Recently, there has been a rise in abuse of synthetic cannabinoids (SCBs). The consumption of SCBs results in various effects and can induce toxic reactions, including paranoia, seizures, tachycardia and even death. 1-Naphthyl 1-(4-fluorobenzyl)-1H-indole-3-carboxylate (FDU-PB-22) is a third generation SCB whose metabolic pathway has not been fully characterized. In this study, we conducted in vitro pharmacokinetic analysis of FDU-PB-22 metabolism. Metabolic reactions containing FDU-PB-22 and human liver microsomes (HLMs) were independent of NADPH but not UDP-glucuronic acid (UDPGA), suggesting that UDP-glucuronosyltransferases (UGTs) are the primary enzymes involved in this metabolism. It was further determined that the metabolite extensively formed after incubating FDU-PB-22 with UDPGA in HLMs was the glucuronide of FDU-PB-22 3-carboxyindole (FBI-COOH). Various hepatic UGTs showed enzymatic activity for FBI-COOH. A series of UGT inhibitors showed moderate to strong inhibition of FBI-COOH-glucuronidation in HLMs, suggesting that multiple UGT isoforms are involved in FBI-COOH-glucuronidation in the liver. Interestingly, an extra-hepatic isoform, UGT1A10, exhibited the highest activity with a K m value of 38 µM and a V max value of 5.90 nmol/min/mg. Collectively, these results suggest that both genetic mutations of and the co-administration of inhibitors for FDU-PB-22-metabolizing UGTs will likely increase the risk of FDU-PB-22-induced toxicity.

Glycyrrhizin, a sweet triterpenoid saponin found in the roots and stolons of Glycyrrhiza species (licorice), is an important active ingredient in traditional herbal medicine. We previously identified two cytochrome P450 monooxygenases, CYP88D6 and CYP72A154, that produce an aglycone of glycyrrhizin, glycyrrhetinic acid, in Glycyrrhiza uralensis. The sugar moiety of glycyrrhizin, which is composed of two glucuronic acids, makes it sweet and reduces its side-effects. Here, we report that UDP-glycosyltransferase (UGT) 73P12 catalyzes the second glucuronosylation as the final step of glycyrrhizin biosynthesis in G. uralensis; the UGT73P12 produced glycyrrhizin by transferring a glucuronosyl moiety of UDP-glucuronic acid to glycyrrhetinic acid 3-O-monoglucuronide. We also obtained a natural variant of UGT73P12 from a glycyrrhizin-deficient (83-555) strain of G. uralensis. The natural variant showed loss of specificity for UDP-glucuronic acid and resulted in the production of an alternative saponin, glucoglycyrrhizin. These results are consistent with the chemical phenotype of the 83-555 strain, and suggest the contribution of UGT73P12 to glycyrrhizin biosynthesis in planta. Furthermore, we identified Arg32 as the essential residue of UGT73P12 that provides high specificity for UDP-glucuronic acid. These results strongly suggest the existence of an electrostatic interaction between the positively charged Arg32 and the negatively charged carboxy group of UDP-glucuronic acid. The functional arginine residue and resultant specificity for UDP-glucuronic acid are unique to UGT73P12 in the UGT73P subfamily. Our findings demonstrate the functional specialization of UGT73P12 for glycyrrhizin biosynthesis during divergent evolution, and provide mechanistic insights into UDP-sugar selectivity for the rational engineering of sweet triterpenoid saponins., (© 2019 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Cancer metastasis is the primary cause of morbidity and mortality, and accounts for up to 95% of cancer-related deaths 1 . Cancer cells often reprogram their metabolism to efficiently support cell proliferation and survival 2,3 . However, whether and how those metabolic alterations contribute to the migration of tumour cells remain largely unknown. UDP-glucose 6-dehydrogenase (UGDH) is a key enzyme in the uronic acid pathway, and converts UDP-glucose to UDP-glucuronic acid 4 . Here we show that, after activation of EGFR, UGDH is phosphorylated at tyrosine 473 in human lung cancer cells. Phosphorylated UGDH interacts with Hu antigen R (HuR) and converts UDP-glucose to UDP-glucuronic acid, which attenuates the UDP-glucose-mediated inhibition of the association of HuR with SNAI1 mRNA and therefore enhances the stability of SNAI1 mRNA. Increased production of SNAIL initiates the epithelial-mesenchymal transition, thus promoting the migration of tumour cells and lung cancer metastasis. In addition, phosphorylation of UGDH at tyrosine 473 correlates with metastatic recurrence and poor prognosis of patients with lung cancer. Our findings reveal a tumour-suppressive role of UDP-glucose in lung cancer metastasis and uncover a mechanism by which UGDH promotes tumour metastasis by increasing the stability of SNAI1 mRNA.

Neonates experience dramatic changes in the disposition of drugs after birth as a result of enzyme maturation and environmental adjustment, challenging therapeutic decision making. In this research, we establish postnatal age, postmenstrual age, and body weight as physiologically reasonable predictors of morphine's clearance in neonates. By integrating knowledge of bilirubin, morphine, and other drugs metabolized by glucuronidation pathways from previously published studies, we hypothesize that uridine diphosphate glucuronic acid, a postnatal age-dependent sugar, plays an important role in the metabolism of morphine during the first week of life. This finding can be extended to other drugs metabolized by uridine diphosphate glucuronosyltransferase pathways in neonates and thus has important clinical implications for the use of drugs in this population., (© 2019 The Authors CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

Accumulating evidence indicates that several human UDP-glucuronosyltransferase (UGT) enzymes catalyze both glucuronidation and glucosidation reactions. Baculovirus-infected insect cells [ Trichoplusia ni and Spodoptera frugiperda (Sf9)] are used widely for the expression of recombinant human UGT enzymes. Following the observation that control Supersomes (c-SUP) express a native enzyme capable of glucosidating morphine, we characterized the glucosidation of a series of aglycones with a hydroxyl (aliphatic or phenolic), carboxylic acid, or amine functional group by c-SUP and membranes from uninfected Sf9 cells. Although both enzyme sources glucosidated the phenolic substrates investigated, albeit with differing activities, differences were observed in the selectivities of the native UDP-glucosyltransferases toward aliphatic alcohols, carboxylic acids, and amines. For example, zidovudine was solely glucosidated by c-SUP. By contrast, c-SUP lacked activity toward the amines lamotrigine and trifluoperazine and did not form the acyl glucoside of mycophenolic acid, reactions all catalyzed by uninfected Sf9 membranes. Glucosidation intrinsic clearances were high for several substrates, notably 1-hydroxypyrene (∼1400-1900 µ l/min⋅mg). The results underscore the importance of including control cell membranes in the investigation of drug and chemical glucosidation by UGT enzymes expressed in T. ni (High-Five) and Sf9 cells. In a coincident study, we observed that UGT1A5 expressed in Sf9, human embryonic kidney 293T, and COS7 cells lacked glucuronidation activity toward prototypic phenolic substrates. However, Sf9 cells expressing UGT1A5 glucosidated 1-hydroxypyrene with UDP-glucuronic acid as the cofactor, presumably due to the presence of UDP-glucose as an impurity. Artifactual glucosidation may explain, at least in part, a previous report of phenolic glucuronidation by UGT1A5., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

UDP-glucuronosyltransferase (UGT)-mediated metabolism is possibly the most important conjugation reaction for marketed drugs. However, there are currently no generally accepted standard incubation conditions for UGT microsomal assays, and substantial differences in experimental design and methodology between laboratories hinder cross-study comparison of in vitro activities. This study aimed to define optimal experimental conditions to determine glucuronidation activity of multiple UGT isoforms simultaneously using human liver microsomes. Hepatic glucuronidation activities of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B10, UGT2B15, and UGT2B17 were determined using cocktail incubations of 10 UGT probe substrates. Buffer components and cosubstrates were assessed over a range of concentrations including magnesium chloride (MgCl 2 ; 0-10 mM) and uridine 5'-diphosphoglucuronic acid (UDPGA; 1-25 mM) with either Tris-HCl or potassium phosphate buffer (100 mM, pH 7.4). Greater microsomal glucuronidation activity by different hepatic UGT isoforms was obtained using 10 mM MgCl 2 and 5 mM UDPGA with 100 mM Tris-HCl buffer. The influence of bovine serum albumin (BSA; 0.1%-2% w/v) on glucuronidation activity was also assessed. Enzyme- and substrate-dependent effects of BSA were observed, resulting in decreased total activity of UGT1A1, UGT1A3, and UGT2B17 and increased total UGT1A9 and UGT2B7 activity. The inclusion of BSA did not significantly reduce the between-subject variability of UGT activity. Future in vitro UGT profiling studies under the proposed optimized experimental conditions would allow high-quality positive control data to be generated across laboratories, with effective control of a high degree of between-donor variability for UGT activity and for chemical optimization toward lower-clearance drug molecules in a pharmaceutical drug discovery setting., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Solute carrier family 35 member A5 (SLC35A5) is a member of the SLC35A protein subfamily comprising nucleotide sugar transporters. However, the function of SLC35A5 is yet to be experimentally determined. In this study, we inactivated the SLC35A5 gene in the HepG2 cell line to study a potential role of this protein in glycosylation. Introduced modification affected neither N - nor O -glycans. There was also no influence of the gene knock-out on glycolipid synthesis. However, inactivation of the SLC35A5 gene caused a slight increase in the level of chondroitin sulfate proteoglycans. Moreover, inactivation of the SLC35A5 gene resulted in the decrease of the uridine diphosphate (UDP)-glucuronic acid, UDP- N -acetylglucosamine, and UDP- N -acetylgalactosamine Golgi uptake, with no influence on the UDP-galactose transport activity. Further studies demonstrated that SLC35A5 localized exclusively to the Golgi apparatus. Careful insight into the protein sequence revealed that the C-terminus of this protein is extremely acidic and contains distinctive motifs, namely DXEE, DXD, and DXXD. Our studies show that the C-terminus is directed toward the cytosol. We also demonstrated that SLC35A5 formed homomers, as well as heteromers with other members of the SLC35A protein subfamily. In conclusion, the SLC35A5 protein might be a Golgi-resident multiprotein complex member engaged in nucleotide sugar transport.

UDP-glucose dehydrogenase (UGDH) catalyzes the conversion of UDP-glucose to UDP-glucuronic acid by NAD + -dependent two-fold oxidation. Despite extensive investigation into the catalytic mechanism of UGDH, the previously proposed mechanisms regarding the first-step oxidation are somewhat controversial and inconsistent with some biochemical evidence, which instead supports a mechanism involving an NAD + -dependent bimolecular nucleophilic substitution (S N 2) reaction. To verify this speculation, the essential Cys residue of Streptococcus zooepidemicus UGDH ( Sz UGDH) was changed to an Ala residue, and the resulting Cys260Ala mutant and Sz UGDH were then co-expressed in vivo via a single-crossover homologous recombination method. Contrary to the previously proposed mechanisms, which predict the formation of the capsular polysaccharide hyaluronan, the resulting strain instead produced an amide derivative of hyaluronan, as validated via proteinase K digestion, ninhydrin reaction, FT-IR and NMR. This result is compatible with the NAD + -dependent S N 2 mechanism., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Protein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic "lipid-altered" tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Sakuranetin (SKN), found in cherry trees and rice, is a flavanone with various pharmacological activities. It is biosynthesized from naringenin in rice or cherry trees, and the metabolism of SKN has been studied in non-human species. The present study aimed to investigate the metabolic pathways of SKN in human liver microsomes and identify the phase I and phase II metabolites, as well as evaluate the potential for drug⁻herb interactions through the modulation of drug metabolizing enzymes (DMEs). HPLC-DAD and HPLC-electrospray mass spectrometry were used to study the metabolic stability and identify the metabolites from human liver microsomes incubated with SKN. The potential of SKN to inhibit the DMEs was evaluated by monitoring the formation of a DME-specific product. The cytochrome P450 2B6 and 3A4-inductive effects were studied using promoter reporter assays in human hepatocarcinoma cells. The major pathways for SKN metabolism include B-ring hydroxylation, 5- O -demethylation, and conjugation with glutathione or glucuronic acid. The phase I metabolites were identified as naringenin and eriodictyol. SKN was found to be a UDP-glucuronosyltransferases (UGT) 1A9 inhibitor, whereas it induced transactivation of the human pregnane X receptor-mediated cytochrome P450 (CYP) 3A4 gene.

Increased uptake of glucose, a general hallmark of malignant tumors, leads to an accumulation of intermediate metabolites of glycolysis. We investigated whether the high supply of these intermediates promotes their flow into UDP-sugars, and consequently into hyaluronan, a tumor-promoting matrix molecule. We quantified UDP-N-Acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcUA) in human breast cancer biopsies, the levels of enzymes contributing to their synthesis, and their association with the hyaluronan accumulation in the tumor. The content of UDP-GlcUA was 4 times, and that of UDP-GlcNAc 12 times higher in the tumors as compared to normal glandular tissue obtained from breast reductions. The surge of UDP-GlcNAc correlated with an elevated mRNA expression of glutamine-fructose-6-phosphate aminotransferase 2 (GFAT2), one of the key enzymes in the biosynthesis of UDP-GlcNAc, and the expression of GFAT1 was also elevated. The contents of both UDP-sugars strongly correlated with tumor hyaluronan levels. Interestingly, hyaluronan content did not correlate with the mRNA levels of the hyaluronan synthases (HAS1-3), thus emphasizing the role of the UDP-sugar substrates of these enzymes. The UDP-sugars showed a trend to higher levels in ductal vs. lobular cancer subtypes. The results reveal for the first time a dramatic increase of UDP-sugars in breast cancer, and suggest that their high supply drives the accumulation of hyaluronan, a known promoter of breast cancer and other malignancies. In general, the study shows how the disturbed glucose metabolism typical for malignant tumors can influence cancer microenvironment through UDP-sugars and hyaluronan., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Glycans play diverse biological roles, ranging from structural and regulatory functions to mediating cellular interactions. For pathogens, they are also often required for virulence and survival in the host. In Cryptococcus neoformans , an opportunistic pathogen of humans, the acidic monosaccharide glucuronic acid (GlcA) is a critical component of multiple essential glycoconjugates. One of these glycoconjugates is the polysaccharide capsule, a major virulence factor that enables this yeast to modulate the host immune response and resist antimicrobial defenses. This allows cryptococci to colonize the lung and brain, leading to hundreds of thousands of deaths each year worldwide. Synthesis of most glycans, including capsule polysaccharides, occurs in the secretory pathway. However, the activated precursors for this process, nucleotide sugars, are made primarily in the cytosol. This topological problem is resolved by the action of nucleotide sugar transporters (NSTs). We discovered that Uut1 is the sole UDP-GlcA transporter in C. neoformans and is unique among NSTs for its narrow substrate range and high affinity for UDP-GlcA. Mutant cells with UUT1 deleted lack capsule polysaccharides and are highly sensitive to environmental stress. As a result, the deletion mutant is internalized and cleared by phagocytes more readily than wild-type cells are and is completely avirulent in mice. These findings expand our understanding of the requirements for capsule synthesis and cryptococcal virulence and elucidate a critical protein family. IMPORTANCE Cryptococcus neoformans causes lethal meningitis in almost two hundred thousand immunocompromised patients each year. Much of this fungal pathogen's ability to resist host defenses and cause disease is mediated by carbohydrate structures, including a complex polysaccharide capsule around the cell. Like most eukaryotic glycoconjugates, capsule polysaccharides are made within the secretory pathway, although their precursors are generated in the cytosol. Specific transporters are therefore required to convey these raw materials to the site of synthesis. One precursor of particular interest is UDP-glucuronic acid, which donates glucuronic acid to growing capsule polysaccharides. We discovered a highly specific, high-affinity transporter for this molecule. Deletion of the gene encoding this unusual protein abolishes capsule synthesis, alters stress resistance, and eliminates fungal virulence. In this work, we have identified a novel transporter, elucidated capsule synthesis and thereby aspects of fungal pathogenesis, and opened directions for potential antifungal therapy., (Copyright © 2018 Li et al.)

GumK is a membrane-associated glucuronosyltransferase of Xanthomonas campestris that is involved in xanthan gum biosynthesis. GumK belongs to the inverting GT-B superfamily and catalyzes the transfer of a glucuronic acid (GlcA) residue from uridine diphosphate (UDP)-GlcA (UDP-GlcA) to a lipid-PP-trisaccharide embedded in the membrane of the bacteria. The structure of GumK was previously described in its apo- and UDP-bound forms, with no significant conformational differences being observed. Here, we study the behavior of GumK toward its donor substrate UDP-GlcA. Turbidity measurements revealed that the interaction of GumK with UDP-GlcA produces aggregation of protein molecules under specific conditions. Moreover, limited proteolysis assays demonstrated protection of enzymatic digestion when UDP-GlcA is present, and this protection is promoted by substrate binding. Circular dichroism spectroscopy also revealed changes in the GumK tertiary structure after UDP-GlcA addition. According to the obtained emission fluorescence results, we suggest the possibility of exposure of hydrophobic residues upon UDP-GlcA binding. We present in silico-built models of GumK complexed with UDP-GlcA as well as its analogs UDP-glucose and UDP-galacturonic acid. Through molecular dynamics simulations, we also show that a relative movement between the domains appears to be specific and to be triggered by UDP-GlcA. The results presented here strongly suggest that GumK undergoes a conformational change upon donor substrate binding, likely bringing the two Rossmann fold domains closer together and triggering a change in the N-terminal domain, with consequent generation of the acceptor substrate binding site., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: To use permeabilized cells of the fission yeast, Schizosaccharomyces pombe, that expresses human UDP-glucose 6-dehydrogenase (UGDH, EC 1.1.1.22), for the production of UDP-glucuronic acid from UDP-glucose., Results: In cell extracts no activity was detected. Therefore, cells were permeabilized with 0.3 % (v/v) Triton X-100. After washing away all low molecular weight metabolites, the permeabilized cells were directly used as whole cell biocatalyst. Substrates were 5 mM UDP-glucose and 10 mM NAD(+). Divalent cations were not added to the reaction medium as they promoted UDP-glucose hydrolysis. With this reaction system 5 mM UDP-glucose were converted into 5 mM UDP-glucuronic acid within 3 h., Conclusions: Recombinant permeabilized cells of S. pombe can be used to synthesize UDP-glucuronic acid with 100 % yield and selectivity.

UDP-glucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS) are the two enzymes responsible for the biosynthesis of UDP-xylose from UDP-glucose. Several UGDs from bacterial sources, which oxidize UDP-glucose to glucuronic acid, have been found and functionally characterized whereas only few reports on bacterial UXS isoforms exist. Rhodothermus marinus, a halothermophilic bacterium commonly found in hot springs, proved to be a valuable source of carbohydrate active enzymes of biotechnological interest, such as xylanases, mannanases, and epimerases. However, no enzymes of R. marinus involved in the biosynthesis or modification of nucleotide sugars have been reported yet. Herein, we describe the cloning and characterization of two putative UGD (RmUGD1 and RmUGD2) and one UXS (RmUXS) isoform from this organism. All three enzymes could be expressed in recombinant form and purified to near homogeneity. UPLC- and NMR-based activity tests showed that RmUGD1 and RmUXS are indeed active enzymes, whereas no enzymatic activity could be detected by RmUGD2. Both RmUGD1 and RmUXS showed a temperature optimum of 60 °C, with almost no loss of activity after 1 h exposure at 70 °C. No metal ions were required for enzymatic activities. Zn(2+) ions strongly inhibited both enzymes. RmUGD1 showed higher salt tolerance and had a higher pH optimum than RmUXS. Furthermore, RmUGD1 was inhibited by UDP-xylose at higher concentrations. By coupling recombinant RmUXS and RmUGD1, UDP-xylose could be successfully synthesized directly from UDP-glucose. The high activity of the herein described enzymes make RmUGD1 and RmUXS the first thermo-tolerant biocatalysts for the synthesis of UDP-glucuronic acid and UDP-xylose.

Bavachinin (BCI), a major bioactive compound in Chinese herbal Psoralea corylifolia, possesses a wide range of biological activities. In this study, the glucuronidation pathway of BCI was characterized for the first time, by using pooled human liver microsomes (HLM), pooled human intestine microsomes (HIM) and recombinant human UDP-glucosyltransferases (UGTs). One mono-glucuronide was detected in HLM in the presence of uridine-diphosphate glucuronic acid (UDPGA), and it was biosynthesized and well-characterized as BCI-4'-O-glucuronide (BCIG). Reaction phenotyping assay showed that UGT1A1, UGT1A3 and UGT1A8 were involved in BCI-4'-O-glucuronidation, while UGT1A1 and UGT1A8 displayed the higher catalytic ability among all tested UGT isoforms. Kinetic analysis demonstrated that BCI-4'-O-glucuronidation in both HLM and UGT1A1 followed sigmoidal kinetic behaviors and displayed much close Km values (12.4 μM in HLM & 9.7 μM in UGT1A1). Both chemical inhibition assays and correlation analysis demonstrated that UGT1A1 displayed a predominant role in BCI-4'-O-glucuronidation in HLM. Both HIM and UGT1A8 exhibited substrate inhibition at high concentrations, and Km values of HIM and UGT1A8 were 3.6 and 2.3 μM, respectively. Similar catalytic efficiencies were observed for HIM (199.3 μL/min/mg) and UGT1A8 (216.2 μL/min/mg). These findings suggested that UGT1A1 and UGT1A8 were the primary isoforms involved in BCI-4'-O-glucuronidation in HLM, and HIM, respectively., (Copyright © 2015 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

The in vitro metabolism of flavokawains A, B, and C (FKA, FKB, FKC), methoxylated chalcones from Piper methysticum, was examined using human liver microsomes. Phase I metabolism and phase II metabolism (glucuronidation) as well as combined phase I+II metabolism were studied. For identification and structure elucidation of microsomal metabolites, LC-HRESIMS and NMR techniques were applied. Major phase I metabolites were generated by demethylation in position C-4 or C-4' and hydroxylation predominantly in position C-4, yielding FKC as phase I metabolite of FKA and FKB, helichrysetin as metabolite of FKA and FKC, and cardamonin as metabolite of FKC. To an even greater extent, flavokawains were metabolized in the presence of uridine diphosphate (UDP) glucuronic acid by microsomal UDP-glucuronosyl transferases. For all flavokawains, monoglucuronides (FKA-2'-O-glucuronide, FKB-2'-O-glucuronide, FKC-2'-O-glucuronide, FKC-4-O-glucuronide) were found as major phase II metabolites. The dominance of generated glucuronides suggests a role of conjugated chalcones as potential active compounds in vivo.

Objectives: To determine the reaction kinetics for regioselective glucuronidation of gingerols (i.e. 6-, 8- and 10-gingerol) by human liver microsomes and expressed UDP-glucuronosyltransferase (UGT) enzymes, and to identify the main UGT enzymes involved in regioselective glucuronidation of gingerols., Methods: The rates of glucuronidation were determined by incubating the gingerols with uridine diphosphoglucuronic acid-supplemented microsomes. Kinetic parameters were derived by fitting an appropriate model to the data. Activity correlation analyses were performed to identify the main UGT enzymes contributing to hepatic metabolism of gingerols., Key Findings: Glucuronidation at the 4'-OH group was much more favoured than that at 5-OH. The degree of position preference was compound-dependent; the catalytic efficiency ratios of 4'-O- to 5-O-glucuronidation were 9.1, 19.7 and 2.9 for 6-, 8- and 10-gingerol, respectively. UGT1A8 (an intestinal enzyme), UGT1A9 and UGT2B7 were the enzymes showing the highest activity towards gingerols. Formation of 5-O-glucuronide was mainly catalysed by UGT1A9. UGT2B7 was the only enzyme that generated glucuronides at both 4'-OH and 5-OH sites, although a strong position preference was observed with 4'-OH (≥80.2%). Further, activity correlation analyses indicated that UGT2B7 and UGT1A9 were primarily responsible for 4'-O-glucuronidation and 5-O-glucuronidation of gingerols in the liver, respectively., Conclusions: Gingerols were metabolized by multiple hepatic and gastrointestinal UGT enzymes. Also, UGT1A9 and 2B7 were the main contributors to regioselective glucuronidation of gingerols in the liver., (© 2014 Royal Pharmaceutical Society.)

We investigated to compare species differences in amitriptyline (AMI) metabolism among mouse, rat, dog, and human liver microsomes. We developed a method for simultaneous determination of metabolic stability and metabolite profiling using predictive multiple reaction monitoring information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) scanning. In the cofactor-dependent microsomal metabolism study, AMI was metabolized more rapidly in rat and human liver microsomes incubated with NADPH than UDPGA. AMI incubated with NADPH+UDPGA in rat, dog, or mouse liver microsomes disappeared rapidly with a half-life of 3.5, 8.4, or 9.2 min, respectively, but slowly in human liver microsomes with a half-life of 96 min. In total, 9, 10, 11, and 6 putative metabolites of AMI were detected in mouse, rat, dog, and human liver microsomes, respectively, based on mass spectrometric analyses. Kinetic analysis of metabolites in liver microsomes from each species over 120 min showed common metabolic routes of AMI, such as N-demethylation, hydroxylation, and glucuronidation, and subtle interspecies differences in AMI metabolism. The main metabolic routes in mouse, rat, dog, and human liver microsomes were hydroxylation followed by glucuronide conjugation, methyl hydroxylation, and N-demethylation, respectively. The MRM-IDA-EPI method can provide quantitative and qualitative information about metabolic stability and metabolite profiling simultaneously. Moreover, time course analysis of metabolites can not only eliminate false identification of metabolites, but also provide a rationale for proposed metabolic pathways. The MRM-IDA-EPI method combined with time course analysis of metabolites is useful for investigating drug metabolism at the early drug discovery stage., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The bacterial protein ArnA is an essential enzyme in the pathway leading to the modification of lipid A with the pentose sugar 4-amino-4-deoxy-L-arabinose. This modification confers resistance to polymyxins, which are antibiotics that are used as a last resort to treat infections with multiple drug-resistant Gram-negative bacteria. ArnA contains two domains with distinct catalytic functions: a dehydrogenase domain and a transformylase domain. The protein forms homohexamers organized as a dimer of trimers. Here, the crystal structure of apo ArnA is presented and compared with its ATP- and UDP-glucuronic acid-bound counterparts. The comparison reveals major structural rearrangements in the dehydrogenase domain that lead to the formation of a previously unobserved binding pocket at the centre of each ArnA trimer in its apo state. In the crystal structure, this pocket is occupied by a DTT molecule. It is shown that formation of the pocket is linked to a cascade of structural rearrangements that emerge from the NAD(+)-binding site. Based on these findings, a small effector molecule is postulated that binds to the central pocket and modulates the catalytic properties of ArnA. Furthermore, the discovered conformational changes provide a mechanistic explanation for the strong cooperative effect recently reported for the ArnA dehydrogenase function.

This study characterized the kinetics, variability, and factors that affect UDP-glucuronic acid (UDP-GlcUA) uptake by human liver microsomes (HLM). Biphasic kinetics were observed for UDP-GlcUA uptake by HLM. Uptake affinities (assessed as Kd) of the high- and low-affinity components differed by more than an order of magnitude (13 ± 6 vs. 374 ± 175 µM), but were comparable in terms of the maximal rate of uptake, with mean Vmax values differing less than 2.3-fold (56 ± 26 vs. 131 ± 35 pmol/min per mg). Variability in total intrinsic transporter activity (Uint) for microsomal UDP-GlcUA uptake across 12 livers was less than 4-fold. Experiments performed to optimize the conditions for microsomal UDP-GlcUA uptake demonstrated that both components were trans-stimulated by preloading (luminal addition) with an alternate UDP-sugar, and essentially abolished by the thiol-alkylating agent N-ethylmaleimide. Furthermore, interaction studies undertaken with a panel of drugs, alternate UDP-sugars, and glucuronide conjugates, at low (2.5 μM) and high (1000 μM) UDP-GlcUA concentrations, demonstrated that both components were inhibited to varying extents. Notably, the nucleoside analogs zidovudine, stavudine, lamivudine, and acyclovir inhibited both the high- and low- affinity components of microsomal UDP-GlcUA uptake by >45% at an inhibitor concentration of 100 μM. Taken together, these data demonstrate that human liver microsomal UDP-GlcUA uptake involves multiple protein-mediated components, and raises the possibility of impaired in vivo glucuronidation activity resulting from inhibition of UDP-GlcUA uptake into the endoplasmic reticulum membrane by drugs and other compounds., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Recent studies demonstrated that mutations in B3GNT1, an enzyme proposed to be involved in poly-N-acetyllactosamine synthesis, were causal for congenital muscular dystrophy with hypoglycosylation of α-dystroglycan (secondary dystroglycanopathies). Since defects in the O-mannosylation protein glycosylation pathway are primarily responsible for dystroglycanopathies and with no established O-mannose initiated structures containing a β3 linked GlcNAc known, we biochemically interrogated this human enzyme. Here we report this enzyme is not a β-1,3-N-acetylglucosaminyltransferase with catalytic activity towards β-galactose but rather a β-1,4-glucuronyltransferase, designated B4GAT1, towards both α- and β-anomers of xylose. The dual-activity LARGE enzyme is capable of extending products of B4GAT1 and we provide experimental evidence that B4GAT1 is the priming enzyme for LARGE. Our results further define the functional O-mannosylated glycan structure and indicate that B4GAT1 is involved in the initiation of the LARGE-dependent repeating disaccharide that is necessary for extracellular matrix protein binding to O-mannosylated α-dystroglycan that is lacking in secondary dystroglycanopathies.

The zebrafish genome contains a gene superfamily of 40 Ugt genes that can be divided into Ugt1, Ugt2, and Ugt5 families. Because the encoded zebrafish UDP glucuronosyltransferase (UGT) proteins do not display orthologous relationships to any of the mammalian and avian UGT enzymes based on molecular phylogeny, it is difficult to predict their substrate specificity. Here, we mapped their tissue-specific expression patterns. We showed that the zebrafish UGT enzymes can be glycosylated. We determined their substrate specificity and catalytic activity toward diverse aglycone substrates. Specifically, we measured mRNA levels of each of the 40 zebrafish Ugt genes in 11 adult tissues and found that they are expressed in a tissue-specific manner. Moreover, functional analyses with the donor of UDP glucuronic acid (UDPGA) for each of the 40 zebrafish UGT proteins revealed their substrate specificity toward 10 important aglycones. In particular, UGT1A1, UGT1A7, and UGT1B1 displayed good glucuronidation activities toward most phenolic aglycones (4-methylumbelliferone, 4-nitrophenol, 1-naphthol, bisphenol A, and mycophenolic acid) and the two carboxylic acids (bilirubin and diclofenac). Importantly, some members of the UGT5, a novel UGT family identified recently, are capable of glucuronidating multiple aglycones with the donor cofactor of UDPGA. In particular, UGT5A5, UGT5B2, and UGT5E1 glucuronidate phenols and steroids with high specificity toward steroid hormones of estradiol and testosterone and estrogenic alkylphenols 4-tert-octylphenol. These results shed new insights into the mechanisms by which fish species defend themselves against vast numbers of xenobiotics via glucuronidation conjugations and may facilitate the establishment of zebrafish as a model vertebrate in toxicological, developmental, and pathologic studies., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Glycolipids are mainly found in phototrophic organisms (like plants and cyanobacteria), in Gram-positive bacteria, and a few other bacterial phyla. Besides the function as bulk membrane lipids, they often play a role under phosphate deprivation as surrogates for phospholipids. The Gram-negative Agrobacterium tumefaciens accumulates four different glycolipids under phosphate deficiency, including digalactosyl diacylglycerol and glucosylgalactosyl diacylglycerol synthesized by a processive glycosyltransferase. The other two glycolipids have now been identified by mass spectrometry and nuclear magnetic resonance spectroscopy as monoglucosyl diacylglycerol and glucuronosyl diacylglycerol. These two lipids are synthesized by a single promiscuous glycosyltransferase encoded by the ORF atu2297, with UDP-glucose or UDP-glucuronic acid as sugar donors. The transfer of sugars differing in their chemistry is a novel feature not observed before for lipid glycosyltransferases. Furthermore, this enzyme is the first glucuronosyl diacylglycerol synthase isolated. Deletion mutants of Agrobacterium lacking monoglucosyl diacylglycerol and glucuronosyl diacylglycerol or all glycolipids are not impaired in growth or virulence during infection of tobacco leaf discs. Our data suggest that the four glycolipids and the nonphospholipid diacylglyceryl trimethylhomoserine can mutually replace each other during phosphate deprivation. This redundancy of different nonphospholipids may represent an adaptation mechanism to enhance the competitiveness in nature.

Hyaluronan (HA) is a glycosaminoglycan composed by repeating units of D-glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) that is ubiquitously present in the extracellular matrix (ECM) where it has a critical role in the physiology and pathology of several mammalian tissues. HA represents a perfect environment in which cells can migrate and proliferate. Moreover, several receptors can interact with HA at cellular level triggering multiple signal transduction responses. The control of the HA synthesis is therefore critical in ECM assembly and cell biology; in this review we address the metabolic regulation of HA synthesis. In contrast with other glycosaminoglycans, which are synthesized in the Golgi apparatus, HA is produced at the plasma membrane by HA synthases (HAS1-3), which use cytoplasmic UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. UDP-GlcUA and UDP-hexosamine availability is critical for the synthesis of GAGs, which is an energy consuming process. AMP activated protein kinase (AMPK), which is considered a sensor of the energy status of the cell and is activated by low ATP:AMP ratio, leads to the inhibition of HA secretion by HAS2 phosphorylation at threonine 110. However, the most general sensor of cellular nutritional status is the hexosamine biosynthetic pathway that brings to the formation of UDP-GlcNAc and intracellular protein glycosylation by O-linked attachment of the monosaccharide β-N-acetylglucosamine (O-GlcNAcylation) to specific aminoacid residues. Such highly dynamic and ubiquitous protein modification affects serine 221 residue of HAS2 that lead to a dramatic stabilization of the enzyme in the membranes., (© 2013.)

The metabolic characteristics of ligustrazin (TMPz) in liver microsomes were investigated in the present study. The reaction phenotyping of TMPz metabolism was also identified by in vitro assessment using recombinant human cytochrome P450 enzymes (CYP) and UDP glucuronosyltransferases (UGT). TMPz was incubated at 37 degrees C with human (HLM) and rat liver microsomes (RLM) in the presence of different co-factors. The metabolic stability and enzyme kinetics of TMPz were studied by determining its remaining concentrations with a LC-MS/MS method. TMPz was only metabolically eliminated in the microsomes with NADPH or NADPH+UDPGA. In the HLM and RLM with NADPH+UDPGA, t1/2, K(m) and V(max) of TMPz were 94.24 +/- 4.53 and 105.07 +/- 9.44 min, 22.74 +/- 1.89 and 33.09 +/- 2.74 micromol x L(-1), 253.50 +/- 10.06 and 190.40 +/- 8.35 nmol x min(-1) x mg(-1) (protein), respectively. TMPz showed a slightly higher metabolic rate in HLM than that in RLM. Its primary oxidative metabolites, 2-hydroxymethyl-3, 5, 6-trimethylpyrazine (HTMP), could undergo glucuronide conjugation. The CYP reaction phenotyping of TMPz metabolism was identified using a panel of recombinant CYP isoforms (rCYP) and specific CYP inhibitors in HLM. CYP1A2, 2C9 and 3A4 were found to be the major CYP isoforms involved in TMPz metabolism. Their individual contributions were assessed b) using the method of the total normalized rate to be 19.32%, 27.79% and 52.90%, respectively. It was observed that these CYP isoforms mediated the formation of HTMP in rCYP incubation. The UGT reaction phenotyping of HTMP glucuronidation was also investigated preliminarily by using a panel of 6 UGT isoforms (rUGT). UGT1A1, 1A4 and 1A6 were the predominant isoforms mediated the HTMP glucuronidation. The results above indicate that the metabolism of TMPz involves multiple enzymes mediated phase I and phase II reactions.

In animals, the main precursor for glycosaminoglycan and furthermore proteoglycan biosynthesis, like hyaluronic acid, is UDP-glucuronic acid, which is synthesized via the nucleotide sugar oxidation pathway. Mutations in this pathway cause severe developmental defects (deficiency in the initiation of heart valve formation). In plants, UDP-glucuronic acid is synthesized via two independent pathways. Beside the nucleotide sugar oxidation pathway, a second minor route to UDP-glucuronic acid exist termed the myo-inositol oxygenation pathway. Within this myo-inositol is ring cleaved into glucuronic acid, which is subsequently converted to UDP-glucuronic acid by glucuronokinase and UDP-sugar pyrophosphorylase. Here we report on a similar, but bifunctional enzyme from zebrafish (Danio rerio) which has glucuronokinase/putative pyrophosphorylase activity. The enzyme can convert glucuronic acid into UDP-glucuronic acid, required for completion of the alternative pathway to UDP-glucuronic acid via myo-inositol and thus establishes a so far unknown second route to UDP-glucuronic acid in animals. Glucuronokinase from zebrafish is a member of the GHMP-kinase superfamily having unique substrate specificity for glucuronic acid with a Km of 31 ± 8 µM and accepting ATP as the only phosphate donor (Km: 59 ± 9 µM). UDP-glucuronic acid pyrophosphorylase from zebrafish has homology to bacterial nucleotidyltransferases and requires UTP as nucleosid diphosphate donor. Genes for bifunctional glucuronokinase and putative UDP-glucuronic acid pyrophosphorylase are conserved among some groups of lower animals, including fishes, frogs, tunicates, and polychaeta, but are absent from mammals. The existence of a second pathway for UDP-glucuronic acid biosynthesis in zebrafish likely explains some previous contradictory finding in jekyll/ugdh zebrafish developmental mutants, which showed residual glycosaminoglycans and proteoglycans in knockout mutants of UDP-glucose dehydrogenase.

Obovatol, a major constituent of the leaves of Magnolia obovata Thunb, is known to inhibit nuclear factor-κB activity and arachidonic acid-induced platelet aggregation. This study was performed to identify the metabolites of obovatol in human liver microsomes. Human liver microsomes incubated with obovatol in the presence of NADPH and/or UDPGA resulted in the formation of six metabolites, M1-M6. M1 and M2 were identified as hydroxyobovatol, on the basis of liquid chromatography/tandem mass spectrometric (LC-MS/MS) analysis. M1, M2 and obovatol were further metabolized to their glucuronide conjugates, obovatol-glucuronide (M3), obovatol-diglucuronide (M4) and hydroxyobovatol-glucuronide (M5 and M6). The inhibitory potency of obovatol on eight major human P450s was also investigated in human liver microsomes. In these experiments, obovatol strongly inhibited CYP2C19-mediated S-mephenytoin hydroxylase activity with an IC(50) value of 0.8 µM, which could have implications for drug-drug interactions., (Copyright © 2013 John Wiley & Sons, Ltd.)

Objectives: The aim of this work was to identify the uridine glucuronosyltransferase (UGT) isoforms involved in the metabolism of the broad-spectrum antiviral drug arbidol., Methods: A human liver microsome (HLM) incubation system was employed to catalyse the formation of arbidol glucuronide. The glucuronidation activity of commercially recombinant UGT isoforms towards arbidol was screened. A combination of kinetic analysis and chemical inhibition study was used to determine the UGT isoforms involved in arbidol's glucuronidation., Key Findings: The arbidol glucuronide was detected when arbidol was incubated with HLMs in the presence of UDP-glucuronic acid. The Eadie-Hofstee plot showed that glucuronidation of arbidol was best fit to the Michaelis-Menten kinetic model, and K(m) and apparent V(max) were calculated to be 8.0 ± 0.7 μm and 2.03 ± 0.05 nmol/min/mg protein, respectively. Assessment of a panel of recombinant UGT isoforms revealed that UGT1A1, UGT1A3 and UGT1A9 could catalyse the glucuronidation of arbidol. Kinetic analysis and chemical inhibition study demonstrated that UGT1A9 was the predominant UGT isoform involved in arbidol glucuronidation in HLMs., Conclusions: The major contribution of UGT1A9 towards arbidol glucuronidation was demonstrated in this study., (© 2012 The Authors. JPP © 2012. Royal Pharmaceutical Society.)

LARGE (like-glycosyltransferase) and LARGE2 (glycosyltransferase-like 1B (GYLTL1B)) are homologous Golgi glycosyltransferases possessing two catalytic domains with homology to members of glycosyltransferase families GT8 and GT49. Mutations in human and mouse Large result in muscular dystrophy due to underglycosylation of dystroglycan. The systemic function of LARGE2 is unknown, but at a cellular level the enzyme can substitute for LARGE in glycosylating dystroglycan. Here, we show that LARGE2 catalyzes the same glycosylation reaction as LARGE. It is a bifunctional glycosyltransferase using uridine diphosphate (UDP)-xylose (Xyl) and UDP-glucuronic acid (GlcA) as donor sugars to produce a xyloglucuronan with alternating Xyl and GlcA residues.

The presence of bovine serum albumin (BSA) largely modulates the enzyme kinetics parameters of the human UDP-glucuronosyltransferase (UGT) 1A9, increasing both the apparent aglycone substrate affinity of the enzyme and its limiting reaction velocity (Drug Metab Dispos 39:2117-2129, 2011). For a better understanding of the BSA effects and an examination of whether its presence changes the catalytic mechanism, we have studied the enzyme kinetics of 4-methylumbelliferone glucuronidation by UGT1A9 in the presence and absence of 0.1% BSA, using bisubstrate enzyme kinetic experiments, in both the forward and reverse directions, as well as product and dead-end inhibition. The combined results strongly suggest that the reaction mechanism of UGT1A9, and presumably other human UGTs as well, involves the formation of a compulsory-order ternary-complex, with UDP-α-d-glucuronic acid (UDPGA) as the first binding substrate. Based on the enzyme kinetic parameters measured for the forward and reverse reactions, the equilibrium constant of the overall reaction was calculated (Keq = 574) and the relative magnitudes of the reaction rate constants were elucidated. The inclusion of BSA in the bisubstrate kinetic experiments quantitatively changed the apparent enzyme kinetic parameters, presumably by removing internal inhibitors that bind to the binary enzyme-UDPGA (E-UDPGA) complex, as well as to the ternary E-UDPGA-aglycone complex. Nevertheless, the underlying compulsory-order ternary-complex mechanism with UDPGA binding first is the same in both the absence and presence of BSA. The results offer a novel understanding of UGT enzyme kinetic mechanism and BSA effects.

To explain the low bioavailability of sibiricaxanthone F (SF) following oral administration and to predict its possible metabolites in vivo, the in vitro biotransformation of SF and its metabolic stability in intestinal bacteria (BI) were studied. The metabolism of SF and its aglycone (1,3,6-trihydroxy-2,7-dimethoxyxanthone, Xan) was characterized after incubation with human and rat liver microsomes (HLMs and RLMs). The chemical structures of the metabolites were tentatively identified on the basis of their mass profiles using high performance liquid chromatography coupled with an enhanced mass spectrometry-information dependent acquisition-enhanced product ion (EMS-IDA-EPI) scan mode on a hybrid triple quadrupole-linear ion trap mass spectrometer. The metabolic stability of SF in BI was determined using the multiple reaction monitoring (MRM) mode. As a result, five hydrolyzed metabolites (M1-M4 and Xan) were generated stepwise for SF when it was incubated with BI, whereas two demethylated products (M1 and M5) were detected for Xan in BI. The metabolic stability study of SF in BI revealed that the parent compound could be rapidly hydrolyzed by BI and that Xan, the main corresponding metabolite, increased rapidly. In RLMs and HLMs, two hydrolyzed metabolites (M2 and M3) mediated by CYP450 isoenzymes appeared for SF in the presence of an NADPH-regenerating system, whereas no metabolite was detected for Xan under the same conditions. One (M6) and three glucuronidated metabolites (M7-M9) were generated from SF and Xan, respectively, by phase II isoenzymes in liver microsomal proteins in the presence of uridine 5'-diphosphoglucuronic acid (UDPGA). Overall, the present study revealed that extensive first-pass elimination occurred for SF in intestinal bacteria and hepatic subcellular proteins in vitro, which could be one of the main reasons for the low bioavailability of SF; furthermore, the possible SF-related metabolites in rats and humans were elucidated to be mono-glucuronidated derivatives of Xan following oral administration of SF., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)