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

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

Triterpenoid-biosynthetic UDP-glycosyltransferases from plants

Triterpenoid saponins are naturally occurring structurally diverse glycosides of triterpenes that are widely distributed among plant species. Great interest has been expressed by pharmaceutical and agriculture industries for the glycosylation of triterpenes. Such modifications alter their taste and bio-absorbability, affect their intra?/extracellular transport and storage in plants, and induce novel biological activities in the human body. Uridine diphosphate (UDP)-glycosyltransferases (UGTs) catalyze glycosylation using UDP sugar donors. These enzymes belong to a multigene family and recognize diverse natural products, including triterpenes, as the acceptor molecules. For this review, we collected and analyzed all of the UGT sequences found in Arabidopsis thaliana as well as 31 other species of triterpene-producing plants. To identify potential UGTs with novel functions in triterpene glycosylation, we screened and classified those candidates based on similarity with UGTs from Panax ginseng, Glycine max, Medicago truncatula, Saponaria vaccaria, and Barbarea vulgaris that are known to function in glycosylate triterpenes. We highlight recent findings on UGT inducibility by methyl jasmonate, tissue-specific expression, and subcellular localization, while also describing their catalytic activity in terms of regioselectivity for potential key UGTs dedicated to triterpene glycosylation in plants. Discovering these new UGTs expands our capacity to manipulate the biological and physicochemical properties of such valuable molecules.     

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

Human UDP-glucuronosyltransferases (UGT) are the dominant phase II conjugative drug metabolism enzymes that also play a central role in processing a range of endobiotic compounds. UGTs catalyze the covalent addition of glucuronic acid sugar moieties to a host of therapeutics and environmental toxins, as well as to a variety of endogenous steroids and other signaling molecules. We report the 1.8-A resolution apo crystal structure of the UDP-glucuronic acid binding domain of human UGT isoform 2B7 (UGT2B7), which catalyzes the conjugative elimination of opioid, antiviral, and anticancer drugs. This is the first crystal structure of any region of a mammalian UGT drug metabolism enzyme. Designated UGT2B7 mutants at residues predicted to interact with the UDP-glucuronic acid cofactor exhibited significantly impaired catalytic activity, with maximum effects observed for amino acids closest to the glucuronic acid sugar transferred to the acceptor molecule. Homology modeling of UGT2B7 with related plant flavonoid glucosyltransferases indicates human UGTs share a common catalytic mechanism. Point mutations at predicted catalytic residues in UGT2B7 abrogated activity, strongly suggesting human UGTs also utilize a serine hydrolase-like catalytic mechanism to facilitate glucuronic acid transfer.     

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.     

Albumin Supplement Affects the Metabolism and Metabolism‐Related Drug–Drug Interaction of Fenoprofen Enantiomers

The influence of albumin towards the metabolism behavior of fenoprofen enantiomers and relevant drug–drug interaction was investigated in the present study. The metabolic behavior of fenoprofen enantiomers was compared in a phase II metabolic incubation system with and without bovine serum albumin (BSA). BSA supplement increased the binding affinity parameter (Km) of (R)‐fenoprofen towards human liver microsomes (HLMs) from 148.3 to 214.4 μM. In contrast, BSA supplement decreased the Km of (S)‐fenoprofen towards HLMs from 218.2 to 123.5 μM. For maximum reaction velocity (Vmax), the addition of BSA increased the Vmax of (R)‐fenoprofen from 1.3 to 1.6 nmol/min/mg protein. In the contrast, BSA supplement decreased the Vmax value from 3.3 to 1.5 nmol/min/mg protein. Andrographolide–fenoprofen interaction was used as an example to investigate the influence of BSA supplement towards fenoprofen‐relevant drug–drug interaction. The addition of 0.2% BSA in the incubation system significantly decreased the inhibition potential of andrographolide towards (R)‐fenoprofen metabolism (P < 0.001). Different from (R)‐fenoprofen, the addition of BSA significantly increased the inhibition potential of andrographolide towards the metabolism of (S)‐fenoprofen. BSA supplement also changed the inhibition kinetic type and parameter of andrographolide towards the metabolism of (S)‐fenoprofen. In conclusion, albumin supplement changes the metabolic behavior of fenoprofen enantiomers and the fenoprofen–andrographolide interaction. Chirality 27:436–440, 2015. © 2015 Wiley Periodicals, Inc.     

家蚕核型多角体病毒egt基因的分子进化分析

过PCR方法获得家蚕核型多角体病毒（Bombyx mori nuclearpolyhedrosis virus,BmNPV）的蜕皮甾体尿苷二磷酸葡萄糖基转移酶基因（egt）片段,序列分析表明该片段带有EGT的完整ORF,推测的多肽可形成EGT结构域的高级结构。为了研究egt的起源,利用家蚕基因组数据库,电子克隆了多个家蚕尿苷二磷酸葡萄糖醛酸转移酶（UGT）基因,在此基础上进行了进化分析,表明BmNPV的EGT为antennal-enriched型UGT;推测核型多角体病毒（nucleopolyhedrivirus,NPV）和颗粒体病毒（granulovirus,GV）的egt基因在进化上来源于昆虫的UGT基因,但GV的egt基因在进化上的起源可能要早于NPV的egt基因;可能在昆虫祖先种进化形成不同昆虫目的某一时期,杆状病毒的祖先种从昆虫中获得了antennal-enriched型UGT基因,并进化为egt基因。家蚕的部分UGT基因与转座子元件连锁的基因组结构特点反映了杆状病毒的egt基因可能通过转座子的传递而获得。     

The role of UDP-glucose:hydroxycinnamate glucosyltransferases in phenylpropanoid metabolism and the response to UV-B radiation in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Arabidopsis harbors four UDP-glycosyltransferases that convert hydroxycinnamates (HCAs) to 1-O-β-glucose esters, UGT84A1 (encoded by At4g15480), UGT84A2 (At3g21560), UGT84A3 (At4g15490), and UGT84A4 (At4g15500). To elucidate the role of the individual UGT84A enzymes in planta we analyzed gene expression, UGT activities and accumulation of phenylpropanoids in Arabidopsis wild type plants, ugt mutants and overexpressing lines. Individual ugt84A null alleles did not significantly reduce the gross metabolic flux to the accumulating compounds sinapoylcholine (sinapine) in seeds and sinapoylmalate in leaves. For the ugt84A2 mutant, LC/MS analysis revealed minor qualitative and quantitative changes of several HCA choline esters and of disinapoylspermidine in seeds. Overexpression of individual UGT84A genes caused increased enzyme activities but failed to produce significant changes in the pattern of accumulating HCA esters. For UGT84A3, our data tentatively suggest an impact on cell wall-associated 4-coumarate. Exposure of plants to enhanced UV-B radiation induced the UGT84A-encoding genes and led to a transient increase in sinapoylglucose and sinapoylmalate concentrations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

UDP-glucuronosyltransferase 1A7 polymorphisms are associated with liver cirrhosis

Variations in the UDP-glucuronosyltransferase (UGT) 1A7 gene have been found to be related to the development of hepatocellular carcinoma (HCC). Since the pathogenesis of liver cirrhosis is not dissimilar to that of HCC, we hypothesized that UGT1A7 genetic polymorphisms may be associated with liver cirrhosis. PCR-restriction fragment length polymorphism was utilized to determine UGT for 1A7 genotypes for the 159 patients with liver cirrhosis and 263 gender/age matched controls. Simple logistic regression analysis revealed that significant risk factors for liver cirrhosis were (1) hepatitis B virus (HBV) infection, (2) hepatitis C virus (HCV) infection, (3) HBV infection plus HCV infection and (4) low-activity UGT1A7 genotypes. The results of further multivariate logistic regression confirmed these associations. Interaction of low-activity UGT1A7 genotypes and HBV (or HCV) infection produced an additive effect upon the risk for the development of liver cirrhosis [observed odds ratio (OR) (54.59) greater than the expected OR (18.05)]. UGT1A7 low/low genotype was also related to advanced liver cirrhosis (Child-Pugh classes C and/or B) (OR = 7.50, P = 0.009). This study demonstrates the novel findings that carriage of low-activity UGT1A7 genotypes represents a risk factor for the development and functional severity of liver cirrhosis.