Identification of UGT2B9*2 and UGT2B33 isolated from female rhesus monkey liver

Two UDP-glucuronosyltransferases (UGT2B9(*)2 and UGT2B33) have been isolated from female rhesus monkey liver. Microsomal preparations of the cell lines expressing the UGTs catalyzed the glucuronidation of the general substrate 7-hydroxy-4-(trifluoromethyl)coumarin in addition to selected estrogens (beta-estradiol and estriol) and opioids (morphine, naloxone, and naltrexone). UGT2B9(*)2 displayed highest efficiency for beta-estradiol-17-glucuronide production and did not catalyze the glucuronidation of naltrexone. UGT2B33 displayed highest efficiency for estriol and did not catalyze the glucuronidation of beta-estradiol. UGT2B9(*)2 was found also to catalyze the glucuronidation of 4-hydroxyestrone, 16-epiestriol, and hyodeoxycholic acid, while UGT2B33 was capable of conjugating 4-hydroxyestrone, androsterone, diclofenac, and hyodeoxycholic acid. Three glucocorticoids (cortisone, cortisol, and corticosterone) were not substrates for glucuronidation by liver or kidney microsomes or any expressed UGTs. Our current data suggest the use of beta-estradiol-3-glucuronidation, beta-estradiol-17-glucuronidation, and estriol-17-glucuronidation to assay UGT1A01, UGT2B9(*)2, and UGT2B33 activity in rhesus liver microsomes, respectively.     

Differential glucuronidation of bile acids, androgens and estrogens by human UGT1A3 and 2B7.

In this work, UDP-glucuronosyltransferases (UGTs), UGT1A3, 2B7(H268) and 2B7(Y268), stably expressed in human embryonic kidney cells (HK293) were used to assess glucuronidation activities with a variety of steroid hormone and bile acid substrates. The rate of synthesis of carboxyl- and hydroxyl-linked glucuronides was determined under optimal reaction conditions. Expressed UGT1A3 catalyzed bile acid glucuronidation at high rates exclusively at the carboxyl moiety for all compounds tested. In contrast, UGT1A4 catalyzed bile acid glucuronidation at very low rates exclusively at the 3alpha-hydroxyl function. Both UGT2B7 allelic variants glucuronidated the bile acid substrates at both carboxyl and hydroxyl moieties, however, the 3alpha-hydroxyl position was preferentially conjugated compared to the carboxyl function. Similarly, androsterone, a 3alpha-hydroxylated androgenic steroid, was glucuronidated at very high rates by expressed UGT2B7. Of the estrogenic compounds tested, UGT2B7 catalyzed the glucuronidation of estriol at rates comparable to those determined for androsterone. Other structural discrimination was found with UGT2B7 which had activity toward estriol and estradiol exclusively at the 17beta-OH position, yielding the cholestatic steroid D-ring glucuronides.     

Enantioselective Inhibition of Carprofen Towards UDP‐glucuronosyltransferase (UGT) 2B7

UDP‐glucuronosyltransferases (UGTs)‐catalyzed glucuronidation conjugation reaction plays an important role in the elimination of many important clinical drugs and endogenous substances. The present study aims to investigate the enantioselective inhibition of carprofen towards UGT isoforms. In vitro a recombinant UGT isoforms‐catalyzed 4‐methylumbelliferone (4‐MU) glucuronidation incubation mixture was used to screen the inhibition potential of (R)‐carprofen and (S)‐carprofen towards multiple UGT isoforms. The results showed that (S)‐carprofen exhibited stronger inhibition potential than (R)‐carprofen towards UGT2B7. However, no significant difference was observed for the inhibition of (R)‐carprofen and (S)‐carprofen towards other UGT isoforms. Furthermore, the inhibition kinetic behavior was compared for the inhibition of (S)‐carprofen and (R)‐carprofen towards UGT2B7. A Lineweaver–Burk plot showed that both (S)‐carprofen and (R)‐carprofen exhibited competitive inhibition towards UGT2B7‐catalyzed 4‐MU glucuronidation. The inhibition kinetic parameter (K_i) was calculated to be 7.0 μM and 31.1 μM for (S)‐carprofen and (R)‐carprofen, respectively. Based on the standard for drug–drug interaction, the threshold for (S)‐carprofen and (R)‐carprofen to induce a drug–drug interaction is 0.7 μM and 3.1 μM, respectively. In conclusion, enantioselective inhibition of carprofen towards UDP‐glucuronosyltransferase (UGT) 2B7 was demonstrated in the present study. Using the in vitro inhibition kinetic parameter, the concentration threshold of (S)‐carprofen and (R)‐carprofen to possibly induce the drug–drug interaction was obtained. Therefore, clinical monitoring of the plasma concentration of (S)‐carprofen is more important than (R)‐carprofen to avoid a possible drug–drug interaction between carprofen and the drugs mainly undergoing UGT2B7‐catalyzed metabolism. Chirality 27:189–193, 2015. © 2014 Wiley Periodicals, Inc.     

Characterization of a common deletion polymorphism of the UGT2B17 gene linked to UGT2B15

Members of the human UDP-glucuronosyltransferase 2B family are located in a cluster on chromosome 4q13 and code for enzymes whose gene products are responsible for the normal catabolism of steroid hormones. Two members of this family, UGT2B15 and UGT2B17, share over 95% sequence identity. However, UGT2B17 exhibits broader substrate specificity due to a single amino acid difference. Using gene-specific primers to explore the genomic organization of these two genes, it was determined that UGT2B17 is absent in some human DNA samples. The gene-specific primers demonstrated the presence or absence of a 150 kb genomic interval spanning the entire UGT2B17 gene, revealing that UGT2B17 is present in the human genome as a deletion polymorphism linked to UGT2B15. Furthermore, it is shown that the UGT2B17 deletion polymorphism shows Mendelian segregation and allele frequencies that differ between African Americans and Caucasians.     

The in vitro substrate regiospecificity of recombinant UGT85B1, the cyanohydrin glucosyltransferase from Sorghum bicolor

The in vitro substrate specificity of UDP-glucose:p-hydroxymandelonitrile-O-glucosyltransferase from Sorghum bicolor (UGT85B1) was examined using a range of potential acceptor molecules, including cyanohydrins, terpenoids, phenolics, hexanol derivatives and plant hormones. Qualitative enzyme activity assays employing 20 different putative substrates were performed and 15 proved to be glucosylated using recombinant UGT85B1 isolated from Escherichia coli. K(m) and k(cat) values were determined for nine of these substrates including mandelonitrile, geraniol, nerol and beta-citronellol, 2-hydroxy-3-methoxybenzyl alcohol, 1-hexanol, cis-3-hexen-1-ol, 3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol. UGT85B1 has a rather broad substrate specificity in vitro but shows regiospecificity, demanding the presence of a sterically unhindered hydroxyl group e.g. as part of a cyanohydrin function, as a primary alcohol or as a phenolic hydroxyl group and being influenced by the stereochemistry and/or interactive chemistry of the substituents on the hydroxyl-bearing carbon atom.     

Nuclear UDP-glucuronosyltransferases: identification of UGT2B7 and UGT1A6 in human liver nuclear membranes

We have demonstrated the subcellular localization of the human UDP-glucuronosyltransferases (UGTs), UGT2B7 and UGT1A6, in endoplasmic reticulum (ER) and nuclear membrane from human hepatocytes and cell lines, by in situ immunostaining and Western blot. Double immunostaining for UGT2B7 and calnexin, an ER resident protein, showed that UGT2B7 was equally present in ER and nuclear membrane whereas calnexin was present almost exclusively in ER. Immunogold labeling of HK293 cells expressing UGT2B7 established the presence of UGT2B7 in both nuclear membranes. Enzymatic assays with UGT2B7 substrates confirmed the presence of functional UGT2B7 protein in ER, whole nuclei, and both outer and inner nuclear membranes. This study has identified, for the first time, the presence of UGT2B7 and UGT1A6 in the nucleus and of UGT2B7 in the inner and outer nuclear membranes. This localization may play an important functional role within nuclei: protection from toxic compounds and/or control of steady-state concentrations of nuclear receptor ligands.     

Nonsteroidal anti-inflammatory drugs and phenols glucuronidation in Caco-2 cells: identification of the UDP-glucuronosyltransferases UGT1A6, 1A3 and 2B7

Glucuronidation of phenols (1-naphthol, 4-methylumbelliferone) and nonsteroidal anti-inflammatory drugs (NSAIDs) such as ketoprofen, naproxen and carprofen was investigated in human colon carcinoma Caco-2 cell clones. Glucuronidation of these substances was highly effective in microsomes of the clones PD-7 and TC-7, but much lower in the PF-11 clone. The activity increased up to a maximum after 21 days of culture. RT-PCR experiments indicated that the PD-7 and TC-7 clones expressed the UDP-glucuronosyltransferase (UGT) isoforms UGT1A6, UGT1A3 and UGT2B7, which could account for the glucuronidation of phenols and carboxylic acids observed. Beta-naphthoflavone stimulated by 2-fold the enzyme activity toward 1-naphthol in PD-7 and TC-7 clones, but not in PF-11 cells. This increase was parallel to that of the UGT1A6 mRNA level. Glucuronidation of ketoprofen was also sensitive to the inducing effect of beta-naphthoflavone. Actinomycin D and cycloheximide did not affect the induction of UGT1A6 by beta-naphthoflavone, but suppressed that of ketoprofen UGT. The UGT1A3 mRNA content was enhanced by beta-naphthoflavone; by contrast, that of UGT2B7 was insensitive to the inducer. In conclusion, several UGT isoforms of both families 1 and 2, which glucuronidate phenols and carboxylic NSAIDs, have been identified in Caco-2 cells. They are differently sensitive to beta-naphthoflavone.     

Determination of UDP-glucuronosyltransferase UGT2B7 activity in human liver microsomes by ultra-performance liquid chromatography with MS detection

A rapid and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) method was developed for the qualitative and quantitative determination of UGT2B7 activity using 3'-azido-3'-deoxythymidine (AZT) as probe substrate in human liver microsomes (HLMs). The method was validated for the determination of AZT glucuronidation (AZTG) with respect to specificity, linearity, detection limit, recovery, stability, precision and accuracy. The chromatographic separation was achieved on a UPLC BEH C18 column (50 mm x 2.1mm i.d., 1.7 microm), with phase of acetonitrile-water (ratio 6:94). Selective ion reaction (SIR) monitor was specific for AZT, AZTG and I.S. The method was linear over the concentration range 0.5-500 microM for AZTG in spiked HLMs. Good precision and accuracy were obtained for concentrations over the standard curve range. AZTG was stable at 4 degrees C for at least 72 h in spiked liver microsomes samples. The method was successfully used to determine the kinetics of UGT activities toward AZT in HLMs. In addition, the method could determine the effects of fluconazole, a known UGT2B7 selective inhibitor, on AZTG in HLMs. Therefore, this method is suitable for in vitro studies using AZTG formation as an index reaction for UGT2B7 activity.     

Glucuronidation of oxidized fatty acids and prostaglandins B1 and E2 by human hepatic and recombinant UDP-glucuronosyltransferases

Arachidonic acid (AA) can be metabolized to various metabolites, which can act as mediators of cellular processes. The objective of this work was to identify whether AA, prostaglandin (PG) B1 and E2, and 15- and 20-hydroxyeicosatetraenoic acids (15- and 20-HETE) are metabolized via glucuronidation. Assays with human recombinant UDP-glucuronosyltransferase 1A (UGT1A) isoforms revealed that AA and 15-HETE were glucuronidated by UGT1A1, 1A3, 1A4, 1A9, and 1A10, whereas 20-HETE was glucuronidated by UGT1A1 and 1A4 and PGB1 was glucuronidated by UGT1A1, 1A9, and 1A10. All substrates were glucuronidated by recombinant UGT2B7, with AA and 20-HETE being the best substrates. Kinetic analysis of UGT1A1 and 1A9 with AA resulted in Km values of 37.9 and 45.8 microM, respectively. PGB1 was glucuronidated by UGT1A1 with a Km of 26.3 microM. The Km values for all substrates with UGT2B7 were significantly higher than with the UGT1A isoforms. Liquid chromatography-mass spectrometry of glucuronides biosynthesized from PGB1 and 15-HETE showed that hydroxyl groups were the major target of glucuronidation. This work demonstrates a novel metabolic pathway for HETEs and PGs and the role of UGT1A isoforms in this process. These results indicate that glucuronidation may play a significant role in modulation of the availability of these fatty acid derivatives for cellular processes.     

Immunochemical identification of UGT isoforms in human small bowel and in caco-2 cell monolayers

Previous work had suggested the presence of significant levels of UDP-glucuronosyltransferase 1A1 (UGT1A1) catalytic activity in human small intestinal microsomes, with undetectable to low UGT1A6 and 2B7 activities. To confirm the presence of UGT1A1 isoform in human small bowel, to explore the possible absence of UGT1A6 and 2B7 in the organ, and to examine induced Caco-2 cells as a potential model for human intestinal metabolism, Western blot analysis was performed using specific antibodies to the relevant UGT isoforms. Significant expression of UGT1A1 protein was observed in all samples of human small intestinal microsomes, while UGT1A6 expression was undetectable to faint and UGT2B7 immunoreactivity was faint to detectable. Caco-2 cells treated with typical enzyme-inducing agents resulted in low UGT2B7 expression but failed to mimic the UGT1A1 levels found in human small bowel. Further work needs to be performed to develop a comprehensive in vitro model for human small intestinal first-pass metabolism.     

An amino acid at position 142 in nitrilase from Rhodococcus rhodochrous ATCC 33278 determines the substrate specificity for aliphatic and aromatic nitriles

Nitrilase from Rhodococcus rhodochrous ATCC 33278 hydrolyses both aliphatic and aromatic nitriles. Replacing Tyr-142 in the wild-type enzyme with the aromatic amino acid phenylalanine did not alter specificity for either substrate. However, the mutants containing non-polar aliphatic amino acids (alanine, valine and leucine) at position 142 were specific only for aromatic substrates such as benzonitrile, m-tolunitrile and 2-cyanopyridine, and not for aliphatic substrates. These results suggest that the hydrolysis of substrates probably involves the conjugated pi-electron system of the aromatic ring of substrate or Tyr-142 as an electron acceptor. Moreover, the mutants containing charged amino acids such as aspartate, glutamate, arginine and asparagine at position 142 displayed no activity towards any nitrile, possibly owing to the disruption of hydrophobic interactions with substrates. Thus aromaticity of substrate or amino acid at position 142 in R. rhodochrous nitrilase is required for enzyme activity.     

Crystal structure of Medicago truncatula UGT85H2--insights into the structural basis of a multifunctional (iso)flavonoid glycosyltransferase

(Iso)flavonoids are a diverse group of plant secondary metabolites with important effects on plant, animal and human health. They exist in various glycosidic forms. Glycosylation, which may determine their bioactivities and functions, is controlled by specific plant uridine diphosphate glycosyltransferases (UGTs). We describe a new multifunctional (iso)flavonoid glycosyltransferase, UGT85H2, from the model legume Medicago truncatula with activity towards a number of phenylpropanoid-derived natural products including the flavonol kaempferol, the isoflavone biochanin A, and the chalcone isoliquiritigenin. The crystal structure of UGT85H2 has been determined at 2.1 A resolution, and reveals distinct structural features that are different from those of other UGTs and related to the enzyme's functions and substrate specificities. Structural and comparative analyses revealed the putative binding sites for the donor and acceptor substrates that are located in a large cleft formed between the two domains of the enzyme, and indicated that Trp360 may undergo a conformational change after sugar donor binding to the enzyme. UGT85H2 has higher specificity for flavonol than for isoflavone. Further substrate docking combined with enzyme activity assay and kinetic analysis provided structural insights into this substrate specificity and preference.     

Chiral distinction between the enantiomers of bicyclic alcohols by UDP-glucuronosyltransferases 2B7 and 2B17

The stereoselective binding and transformation of optically pure bicyclic alcohols by human UDP-glucuronosyltransferases from subfamily 2B were investigated. The enantiomers of 1-indanol, 1-tetralol, and 1-benzosuberol were synthesized by asymmetric Corey-Bakshi-Shibata reduction and subjected to glucuronidation assays. The alcohols studied were primarily glucuronidated by UGT2B7 and UGT2B17. The catalytic transformation by UGT2B17 was highly stereoselective, favoring conjugation of the (R)-enantiomers. UGT2B7, on the other hand, did not exhibit stereoselectivity toward 1-benzosuberol, the best substrate in this series. To assess binding affinities to the enzymes, the six different compounds were tested for their efficiency as inhibitors of either UGT2B7 or UGT2B17. The results of the latter analyses indicated that the affinities of both enantiomers of each pair towards UGT2B7 and UGT2B17 were of the same order of magnitude. Therefore, the findings of this study suggest that the spatial arrangement of the hydroxy group plays an important role in the glucuronic acid transfer reaction, but not necessarily in substrate binding to the UGTs.     

Isolation and characterization of the human UGT2B15 gene, localized within a cluster of UGT2B genes and pseudogenes on chromosome 4

Glucuronidation is a major pathway of androgen metabolism and is catalyzed by UDP-glucuronosyltransferase (UGT) enzymes. UGT2B15 and UGT2B17 are 95% identical in primary structure, and are expressed in steroid target tissues where they conjugate C19 steroids. Despite the similarities, their regulation of expression are different; however, the promoter region and genomic structure of only the UGT2B17 gene have been characterizedX to date. To isolate the UGT2B15 gene and other novel steroid-conjugating UGT2B genes, eight P-1-derived artificial chromosomes (PAC) clones varying in length from 30 kb to 165 kb were isolated. The entire UGT2B15 gene was isolated and characterized from the PAC clone 21598 of 165 kb. The UGT2B15 and UGT2B17 genes are highly conserved, are both composed of six exons spanning approximately 25 kb, have identical exon sizes and have identical exon-intron boundaries. The homology between the two genes extend into the 5'-flanking region, and contain several conserved putative cis-acting elements including Pbx-1, C/EBP, AP-1, Oct-1 and NF/kappaB. However, transfection studies revealed differences in basal promoter activity between the two genes, which correspond to regions containing non-conserved potential elements. The high degree of homology in the 5'-flanking region between the two genes is lost upstream of -1662 in UGT2B15, and suggests a site of genetic recombination involved in duplication of UGT2B genes. Fluorescence in situ hybridization mapped the UGT2B15 gene to chromosome 4q13.3-21.1. The other PAC clones isolated contain exons from the UGT2B4, UGT2B11 and UGT2B17 genes. Five novel exons, which are highly homologous to the exon 1 of known UGT2B genes, were also identified; however, these exons contain premature stop codons and represent the first recognized pseudogenes of the UGT2B family. The localization of highly homologous UGT2B genes and pseudogenes as a cluster on chromosome 4q13 reveals the complex nature of this gene locus, and other novel homologous UGT2B genes encoding steroid conjugating enzymes are likely to be found in this region of the genome.     

Glucuronidation of linoleic acid diols by human microsomal and recombinant UDP-glucuronosyltransferases: identification of UGT2B7 as the major isoform involved

Recent reports suggest that linoleic acid (LA) epoxides and diols are associated with important physiological, pharmacological, and pathological events in vivo. We have shown recently that LA-diols are excellent substrates for human liver microsomal UDP-glucuronosyltransferases (UGTs); however, it is not known if other human tissues glucuronidate LA-diols or which UGT isozyme(s) is involved. The present studies with human intestinal microsomes indicate that glucuronidation of LA-diols occurs throughout the gastrointestinal tract, with the highest activity in the small intestine. LA-diols yielded exclusively hydroxyl-linked glucuronides, whereas LA yielded the carboxyl-linked glucuronide. Studies with human recombinant UGTs demonstrated that only UGT2B7 glucuronidated LA and LA-diols. Kinetic analysis with UGT2B7 yielded apparent K(m) values in the range of 40-70 microM and V(max) values from 4.5 to 5.4 nmol/mg x min. These studies indicate that LA and LA-diols are excellent substrates for intestinal UGTs and provide the first evidence for UGT2B7 being the major isoform involved.     

Peroxisome proliferator-activated receptor alpha induces hepatic expression of the human bile acid glucuronidating UDP-glucuronosyltransferase 2B4 enzyme

Glucuronidation, a major metabolic pathway for a large variety of endobiotics and xenobiotics, is catalyzed by enzymes belonging to the UDP-glucuronosyltransferase (UGT) family. Among UGT enzymes, UGT2B4 conjugates a large variety of endogenous and exogenous molecules and is considered to be the major bile acid conjugating UGT enzyme in human liver. In the present study, we identify UGT2B4 as a novel target gene of the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR alpha), which mediates the hypolipidemic action of fibrates. Incubation of human hepatocytes or hepatoblastoma HepG2 and Huh7 cells with synthetic PPAR alpha agonists, fenofibric acid, or Wy 14643 resulted in an increase of UGT2B4 mRNA levels. Furthermore, treatment of HepG2 cells with Wy 14643 induced the glucuronidation of hyodeoxycholic acid, a specific bile acid UGT2B4 substrate. Analysis of UGT2B mRNA and protein levels in PPAR alpha wild type and null mice revealed that PPAR alpha regulates both basal and fibrate-induced expression of these enzymes in rodents also. Finally, a PPAR response element was identified in the UGT2B4 promoter by site-directed mutagenesis and electromobility shift assays. These results demonstrate that PPAR alpha agonists may control the catabolism of cytotoxic bile acids and reinforce recent data indicating that PPAR alpha, which has been largely implicated in the control of lipid and cholesterol metabolism, is also an important modulator of the metabolism of endobiotics and xenobiotics in human hepatocytes.     

Kinetic analysis of phenylalanine dehydrogenase mutants designed for aliphatic amino acid dehydrogenase activity with guidance from homology-based modelling.

Through comparison with the high-resolution structure of Clostridium symbiosum glutamate dehydrogenase, the different substrate specificities of the homologous enzymes phenylalanine dehydrogenase and leucine dehydrogenase were attributed to two residues, glycine 124 and leucine 307, in Bacillus sphaericus phenylalanine dehydrogenase, which are replaced with alanine and valine in leucine dehydrogenases. As predicted, making these substitutions in phenylalanine dehydrogenase decreased the specific activity towards aromatic substrates and enhanced the activity towards some aliphatic amino acids in standard assays with fixed concentrations of both substrates. This study did not, however, distinguish effects on affinity from those on maximum catalytic rate. A fuller kinetic characterization of the single- and double-mutant enzymes now reveals that the extent of the shift in specificity was underestimated in the earlier study. The maximum catalytic rates for aromatic substrates are reduced for all the mutants, but, in addition, the apparent Km values are higher for the single-mutant G124A and double-mutant G124A/L307V compared with the wild-type enzyme. Conversely, specificity constants (kcat/Km) for the nonpolar aliphatic amino acids and the corresponding 2-oxoacids for the mutants are all markedly higher than for the wild type, with up to a 40-fold increase for l-norvaline and a 100-fold increase for its 2-oxoacid in the double mutant. In some cases a favourable change in Km was found to outweigh a smaller negative change in kcat. These results emphasize the risk of misjudging the outcome of protein engineering experiments through too superficial an analysis. Overall, however, the success of the predictions from molecular modelling indicates the usefulness of this strategy for engineering new specificities, even in advance of more detailed 3D structural information.     

Chirality Influence of Zaltoprofen Towards UDP‐Glucuronosyltransferases (UGTs) Inhibition Potential

Zaltoprofen (ZLT) is a nonsteroidal antiinflammation drug, and has been clinically employed to treat rheumatoid arthritis, osteoarthritis, and other chronic inflammatory pain conditions. The present study aims to investigate the chirality influence of zaltoprofen towards the inhibition potential towards UDP‐glucuronosyltransferases (UGTs) isoforms. In vitro a recombinant UGT isoforms‐catalyzed 4‐methylumbelliferone (4‐MU) glucuronidation incubation system was employed to investigate the inhibition of (R)‐zaltoprofen and (S)‐zaltoprofen towards UGT isoforms. The inhibition difference capability was observed for the inhibition of (R)‐zaltoprofen and (S)‐zaltoprofen towards UGT1A8 and UGT2B7, but not for other tested UGT isoforms. (R)‐zaltoprofen exhibited noncompetitive inhibition towards UGT1A8 and competitive inhibition towards UGT2B7. The inhibition kinetic parameters were calculated to be 35.3 μM and 19.2 μM for UGT1A8 and UGT2B7. (R)‐zaltoprofen and (S)‐zaltoprofen exhibited a different inhibition type towards UGT1A7. Based on the reported maximum plasma concentration of (R)‐zaltoprofen in vivo, a high drug–drug interaction between (R)‐zaltoprofen and the drugs mainly undergoing UGT1A7, UGT1A8, and UGT2B7‐catalyzed glucuronidation was indicated. Chirality 27:359–363, 2015. © 2015 Wiley Periodicals, Inc.     

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.