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1.  Galactose Starvation in a Bloodstream Form Trypanosoma brucei UDP-Glucose 4′-Epimerase Conditional Null Mutant 
Eukaryotic Cell  2006;5(11):1906-1913.
Galactose metabolism is essential for the survival of Trypanosoma brucei, the etiological agent of African sleeping sickness. T. brucei hexose transporters are unable to transport galactose, which is instead obtained through the epimerization of UDP-glucose to UDP-galactose catalyzed by UDP-glucose 4′-epimerase (galE). Here, we have characterized the phenotype of a bloodstream form T. brucei galE conditional null mutant under nonpermissive conditions that induced galactose starvation. Cellular levels of UDP-galactose dropped rapidly upon induction of galactose starvation, reaching undetectable levels after 72 h. Analysis of extracted glycoproteins by ricin and tomato lectin blotting showed that terminal β-d-galactose was virtually eliminated and poly-N-acetyllactosamine structures were substantially reduced. Mass spectrometric analysis of variant surface glycoprotein confirmed complete loss of galactose from the glycosylphosphatidylinositol anchor. After 96 h, cell division ceased, and electron microscopy revealed that the cells had adopted a morphologically distinct stumpy-like form, concurrent with the appearance of aberrant vesicles close to the flagellar pocket. These data demonstrate that the UDP-glucose 4′-epimerase is essential for the production of UDP-galactose required for galactosylation of glycoproteins and that galactosylation of one or more glycoproteins, most likely in the lysosomal/endosomal system, is essential for the survival of bloodstream form T. brucei.
PMCID: PMC1694802  PMID: 17093269
2.  Preliminary X-ray crystallographic studies of UDP-­glucose-4-epimerase from Aspergillus nidulans  
UDP-glucose-4-epimerase from A. nidulans has been crystallized as a complex with the substrate UDP-galactose using the microbatch method under paraffin oil.
UDP-glucose-4-epimerase (GALE) from Aspergillus nidulans was over­expressed in Escherichia coli, purified via His-tag affinity chromatography and cocrystallized with UDP-galactose using the microbatch method. The crystals diffracted to 2.4 Å resolution using synchrotron radiation on the Canadian Light Source 08ID-1 beamline. Examination of the data with d*TREK revealed nonmerohedral twinning, from which a single lattice was ultimately extracted for processing. The final space group was found to be C2, with unit-cell parameters a = 66.13, b = 119.15, c = 161.42 Å, β = 98.48°. An initial structure solution has been obtained via molecular replacement employing human GALE (PDB entry 1hzj) as a template model.
PMCID: PMC3151119  PMID: 21821886
epimerases; galactose metabolism; short-chain dehydrogenases; Leloir pathway
Mammalian 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD) is a member of the short chain dehydrogenase/reductase. It is a key steroidogenic enzyme that catalyzes the first step of the multienzyme pathway conversion of circulating dehydroepiandrosterone and pregnenolone to active steroid hormones. A three dimensional model of a ternary complex of human 3β-HSD type 1 (3β-HSD_1) with an NAD cofactor and androstenedione product has been developed based upon X-ray structures of the ternary complex of E. coli UDP-galactose 4-epimerase (UDPGE) with an NAD cofactor and substrate (PDB_AC: 1NAH) and the ternary complex of human type 1 17β-hydroxysteroid dehydrogenase (17β-HSD_1) with an NADP cofactor and androstenedione (PDB_AC: 1QYX). The dimeric structure of the enzyme was built from two monomer models of 3β-HSD_1 by respective 3D superposition with A and B subunits of the dimeric structure of Streptococcus suis DTDP-D-glucose 4,6-dehydratase (PDB_AC: 1KEP). The 3D model structure of 3β-HSD_1 has been successfully used for the rational design of mutagenic experiments to further elucidate the key substrate binding residues in the active site as well as the basis for dual function of the 3β-HSD_1 enzyme. The structure based mutant enzymes, Asn100Ser, Asn100Ala, Glu126Leu, His232Ala, Ser322Ala and Asn323Leu, have been constructed and functionally characterized. The mutagenic experiments have confirmed the predicted roles of the His232 and Asn323 residues in recognition of the 17-keto group of the substrate and identified Asn100 and Glu126 residues as key residues that participate for the dehydrogenase and isomerization reactions respectively.
PMCID: PMC1971842  PMID: 16889958
3β-hydroxysteroid dehydrogenase; short-chain oxidoreductase; 3D model structure; bioinformatics; rational proteomics; structure based mutagenesis; structure-function relationship
4.  Identification, subcellular localization, biochemical properties, and high-resolution crystal structure of Trypanosoma brucei UDP-glucose pyrophosphorylase 
Glycobiology  2010;20(12):1619-1630.
The protozoan parasite Trypanosoma brucei is the causative agent of the cattle disease Nagana and human African sleeping sickness. Glycoproteins play key roles in the parasite’s survival and infectivity, and the de novo biosyntheses of the sugar nucleotides UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine, and GDP-fucose have been shown to be essential for their growth. The only route to UDP-Gal in T. brucei is through the epimerization of UDP-glucose (UDP-Glc) by UDP-Glc 4′-epimerase. UDP-Glc is also the glucosyl donor for the unfolded glycoprotein glucosyltransferase (UGGT) involved in glycoprotein quality control in the endoplasmic reticulum and is the presumed donor for the synthesis of base J (β-d-glucosylhydroxymethyluracil), a rare deoxynucleotide found in telomere-proximal DNA in the bloodstream form of T. brucei. Considering that UDP-Glc plays such a central role in carbohydrate metabolism, we decided to characterize UDP-Glc biosynthesis in T. brucei. We identified and characterized the parasite UDP-glucose pyrophosphorylase (TbUGP), responsible for the formation of UDP-Glc from glucose-1-phosphate and UTP, and localized the enzyme to the peroxisome-like glycosome organelles of the parasite. Recombinant TbUGP was shown to be enzymatically active and specific for glucose-1-phosphate. The high-resolution crystal structure was also solved, providing a framework for the design of potential inhibitors against the parasite enzyme.
PMCID: PMC3270307  PMID: 20724435
kinetoplastids; sugar nucleotide metabolism; Trypanosoma brucei; UDP-glucose; UDP-glucose pyrophosphorylase
5.  The Molecular Dynamics of Trypanosoma brucei UDP-Galactose 4′-Epimerase: A Drug Target for African Sleeping Sickness 
Chemical Biology & Drug Design  2012;80(2):173-181.
During the past century, several epidemics of human African trypanosomiasis, a deadly disease caused by the protist Trypanosoma brucei, have afflicted sub-Saharan Africa. Over 10 000 new victims are reported each year, with hundreds of thousands more at risk. As current drug treatments are either highly toxic or ineffective, novel trypanocides are urgently needed. The T. brucei galactose synthesis pathway is one potential therapeutic target. Although galactose is essential for T. brucei survival, the parasite lacks the transporters required to intake galactose from the environment. UDP-galactose 4′-epimerase (TbGalE) is responsible for the epimerization of UDP-glucose to UDP-galactose and is therefore of great interest to medicinal chemists. Using molecular dynamics simulations, we investigate the atomistic motions of TbGalE in both the apo and holo states. The sampled conformations and protein dynamics depend not only on the presence of a UDP-sugar ligand, but also on the chirality of the UDP-sugar C4 atom. This dependence provides important insights into TbGalE function and may help guide future computer-aided drug discovery efforts targeting this protein.
PMCID: PMC3399956  PMID: 22487100
African sleeping sickness; molecular dynamics; protein structure; TbGalE; Trypanosoma brucei; UDP-Galactose-4′-Epimerase
6.  The Molecular Dynamics of Trypanosoma brucei UDP-Galactose 4′-Epimerase: A Drug Target for African Sleeping Sickness 
Chemical biology & drug design  2012;80(2):173-181.
During the past century, several epidemics of human African trypanosomiasis, a deadly disease caused by the protist Trypanosoma brucei, have afflicted sub-Saharan Africa. Over 10,000 new victims are reported each year, with hundreds of thousands more at risk. As current drug treatments are either highly toxic or ineffective, novel trypanocides are urgently needed. The T. brucei galactose-synthesis pathway is one potential therapeutic target. Though galactose is essential for T. brucei survival, the parasite lacks the transporters required to intake galactose from the environment. UDP-galactose 4′-epimerase (TbGalE) is responsible for the epimerization of UDP-glucose to UDP-galactose and so is of great interest to medicinal chemists. Using molecular dynamics simulations, we investigate the atomistic motions of TbGalE in both the apo and holo states. The sampled conformations and protein dynamics depend not only on the presence of a UDP-sugar ligand, but also on the chirality of the UDP-sugar C4 atom. This dependence provides important insights into TbGalE function and may help guide future computer-aided drug-discovery efforts targeting this protein.
PMCID: PMC3399956  PMID: 22487100
TbGalE; Trypanosoma brucei; UDP-Galactose-4’-Epimerase; African Sleeping Sickness; Molecular Dynamics; Protein Structure
7.  UDP-Galactose 4′-Epimerase Activities toward UDP-Gal and UDP-GalNAc Play Different Roles in the Development of Drosophila melanogaster 
PLoS Genetics  2012;8(5):e1002721.
In both humans and Drosophila melanogaster, UDP-galactose 4′-epimerase (GALE) catalyzes two distinct reactions, interconverting UDP-galactose (UDP-gal) and UDP-glucose (UDP-glc) in the final step of the Leloir pathway of galactose metabolism, and also interconverting UDP-N-acetylgalactosamine (UDP-galNAc) and UDP-N-acetylglucosamine (UDP-glcNAc). All four of these UDP-sugars serve as vital substrates for glycosylation in metazoans. Partial loss of GALE in humans results in the spectrum disorder epimerase deficiency galactosemia; partial loss of GALE in Drosophila melanogaster also results in galactose-sensitivity, and complete loss in Drosophila is embryonic lethal. However, whether these outcomes in both humans and flies result from loss of one GALE activity, the other, or both has remained unknown. To address this question, we uncoupled the two activities in a Drosophila model, effectively replacing the endogenous dGALE with prokaryotic transgenes, one of which (Escherichia coli GALE) efficiently interconverts only UDP-gal/UDP-glc, and the other of which (Plesiomonas shigelloides wbgU) efficiently interconverts only UDP-galNAc/UDP-glcNAc. Our results demonstrate that both UDP-gal and UDP-galNAc activities of dGALE are required for Drosophila survival, although distinct roles for each activity can be seen in specific windows of developmental time or in response to a galactose challenge. By extension, these data also suggest that both activities might play distinct and essential roles in humans.
Author Summary
In this manuscript we apply a fruit fly model to explore the relative contributions of each of two different activities attributed to a single enzyme—UDP-galactose 4′-epimerase (GALE); partial impairment of human GALE results in the potentially severe metabolic disorder epimerase deficiency galactosemia. One GALE activity involves interconverting UDP-galactose and UDP-glucose in the Leloir pathway of galactose metabolism; the other activity involves interconverting UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. We have previously demonstrated that complete loss of GALE is embryonic lethal in fruit flies, but it was unclear which GALE activity loss was responsible for the outcome. Using genetically modified fruit flies, we were able to remove or give back each GALE activity individually at different times in development and observe the consequences. Our results demonstrate that both GALE activities are essential, although they play different roles at different times in development. These results provide insight into the normal functions of GALE and also have implications for diagnosis and intervention in epimerase deficiency galactosemia.
PMCID: PMC3359975  PMID: 22654673
8.  UDP-xylose and UDP-galactose synthesis in Trichomonas vaginalis 
Graphical abstract
The genes encoding the enzymes required for UDP-xylose and UDP-galactose synthesis in Trichomonas vaginalis have been identified and the products of the recombinant enzymes analysed.
► Xylose and galactose are components of Trichomonas vaginalis glycans. ► T. vaginalis UDP-xylose synthase and UDP-galactose epimerase genes identified. ► Enzymes were expressed in recombinant form, purified and assayed.
The presence of xylose and galactose residues in the structure of trichomonad lipoglycans was indicated by previous studies and the modification of any glycoconjugate with either monosaccharide requires the respective presence of the nucleotide sugars, UDP-xylose and UDP-galactose. Biosynthesis of UDP-xylose de novo is mediated by UDP-xylose synthase (UXS; UDP-glucuronic acid decarboxylase), which converts UDP-glucuronic acid to UDP-xylose, whereas UDP-galactose can be generated from UDP-glucose by UDP-galactose epimerases (GalE). Trichomonas vaginalis cDNAs, encoding proteins with homology to these enzymes from other eukaryotes, were isolated. The recombinant T. vaginalis UDP-xylose synthase and UDP-galactose epimerase were expressed in Escherichia coli and tested via high pressure liquid chromatography to demonstrate their enzymatic activities. Thereby, in this first report on enzymes involved in glycoconjugate biosynthesis in this organism, we demonstrate the existence of xylose and galactose synthesising pathways in T. vaginalis.
PMCID: PMC3223521  PMID: 22008417
GalE, UDP-galactose-4′-epimerase; UDP-GlcA, UDP-glucuronic acid; UXS, UDP-xylose synthase; UDP-xylose; UDP-galactose; Trichomonas vaginalis
9.  Insights into role of the hydrogen bond networks in substrate recognition by UDP-GalNAc 4-epimerases 
UDP-hexose 4-epimerases are critical in galactose metabolism and often important in lipopolysaccharide biosynthesis as well. Three groups of these enzymes have been reported based on their substrate specificity towards non-acetylated substrates (group 1), dual specificity towards N-acetylated and non-acetylated substrates (group 2) and specificity towards N-acetylated substrates (group 3). We recently reported the structure of a novel UDP-GalNAc 4-epimerase called WbgU and based on the structure proposed a model of specific substrate recognition by UDP-GalNAc 4-epimerases. In this work, we present an analysis of the proposed model of substrate recognition using site-directed mutagenesis of WbgU and crystal structure of the His305Ala mutant. This investigation reveals that the wild-type activity of WbgU is retained in most single-point mutants targeting the active site. However, a graded loss in activity is observed in double-and triple-point mutants with the quadruple-point mutant being completely inactive corroborating the proposed rationale of substrate recognition. Furthermore, crystal structure of the His305Ala mutant shows that the structure is significantly similar to the wild-type WbgU, albeit a loss in the critical hydrogen bond network seated at His305 and ensuing minor conformational changes. It is inferred that the specific and non-specific interactions throughout the active site confer it sufficient elasticity to sustain wild-type activity for several of the single-point mutations.
PMCID: PMC3441825  PMID: 21810411
Lipopolysaccharide; N-acetylglucosamine; Rossmann fold; 4-Epimerase; Galactose metabolism
10.  Computer-Aided Identification of Trypanosoma brucei Uridine Diphosphate Galactose 4′-Epimerase Inhibitors: Toward the Development of Novel Therapies for African Sleeping Sickness 
Journal of Medicinal Chemistry  2010;53(13):5025-5032.
Trypanosoma brucei, the causative agent of human African trypanosomiasis, affects tens of thousands of sub-Saharan Africans. As current therapeutics are inadequate due to toxic side effects, drug resistance, and limited effectiveness, novel therapies are urgently needed. UDP-galactose 4′-epimerase (TbGalE), an enzyme of the Leloir pathway of galactose metabolism, is one promising T. brucei drug target. We here use the relaxed complex scheme, an advanced computer-docking methodology that accounts for full protein flexibility, to identify inhibitors of TbGalE. An initial hit rate of 62% was obtained at 100 μM, ultimately leading to the identification of 14 low-micromolar inhibitors. Thirteen of these inhibitors belong to a distinct series with a conserved binding motif that may prove useful in future drug design and optimization.
PMCID: PMC2895357  PMID: 20527952
11.  Mapping the UDP-Glucuronic Acid Binding Site in UDP-Glucuronosyltransferase-1 A10 by Homology-based Modeling: Confirmation with Biochemical Evidence† 
Biochemistry  2008;47(28):7385-7392.
The UDP-glucuronosyltransferase (UGT) isozyme system is critical for protecting the body against endogenous and exogenous chemicals by linking glucuronic acid donated by UDP-glucuronic acid to a lipophilic acceptor substrate. UGTs convert metabolites, dietary constituents and environmental toxicants to highly excretable glucuronides. Because of difficulties associated with purifying endoplasmic reticulum-bound UGTs for structural studies, we carried out homology-based computer modeling to aid analysis. The search found structural homology in Escherichia coli UDP-galactose 4-epimerase. Consistent with predicted similarities involving the common UDP-moiety in substrates, UDP-glucose and UDP-hexanol amine caused competitive inhibition by Lineweaver-Burk plots. Among predicted binding sites N292, K314, K315 and K404 in UGT1A10, two informative sets of mutants K314R/Q/A/E /G and K404R/E had null activities or 2.7-fold higher/50% less activity, respectively. Scatchard analysis of binding data of affinity-ligand, 5-azido-uridine-[β-32P]-diphosphoglucuronic acid, to purified UGT1A10-His or UGT1A7-His revealed high and low affinity binding sites. 2-Nitro 5-thiocyanobenzoic acid-digested UGT1A10-His bound with radiolabeled affinity-ligand revealed an 11.3- and 14.3-kDa peptide associated with K314 and K404, respectively, in a discontinuous SDS-PAGE system. Similar treatment of 1A10His-K314A bound with the ligand lacked both peptides; 1A10-HisK404R- and 1A10-HisK404E showed 1.3-fold greater- and 50% less-label in the 14.3-kDa peptide, respectively, compared to 1A10-His without affecting the 11.3-kDa peptide. Scatchard analysis of binding data of affinity-ligand to 1A10His-K404R and -K404E showed a 6-fold reduction and a large increase in Kd, respectively. Our results indicate: K314 and K404 are required UDP-glcA binding sites in 1A10, that K404 controls activity and high affinity sites and that K314 and K404 are strictly conserved in 70 aligned UGTs, except for S321--equivalent to K314-- in UGT2B15 and 2B17 and I321 in the inactive UGT8, which suggests UGT2B15 and 2B17 contain suboptimal activity. Hence our data strongly support UDPglcA binding to K314 and K404 in UGT1A10.
PMCID: PMC2587317  PMID: 18570380
12.  Role of Gne and GalE in the Virulence of Aeromonas hydrophila Serotype O34▿  
Journal of Bacteriology  2006;189(2):540-550.
The mesophilic Aeromonas hydrophila AH-3 (serotype O34) strain shows two different UDP-hexose epimerases in its genome: GalE (EC and Gne (EC Similar homologues were detected in the different mesophilic Aeromonas strains tested. GalE shows only UDP-galactose 4-epimerase activity, while Gne is able to perform a dual activity (mainly UDP-N-acetyl galactosamine 4-epimerase and also UDP-galactose 4-epimerase). We studied the activities in vitro of both epimerases and also in vivo through the lipopolysaccharide (LPS) structure of A. hydrophila gne mutants, A. hydrophila galE mutants, A. hydrophila galE-gne double mutants, and independently complemented mutants with both genes. Furthermore, the enzymatic activity in vivo, which renders different LPS structures on the mentioned A. hydrophila mutant strains or the complemented mutants, allowed us to confirm a clear relationship between the virulence of these strains and the presence/absence of the O34 antigen LPS.
PMCID: PMC1797372  PMID: 17098903
13.  Elucidation of Substrate Specificity in Aspergillus nidulans UDP-Galactose-4-Epimerase 
PLoS ONE  2013;8(10):e76803.
The frequency of invasive fungal infections has rapidly increased in recent years. Current clinical treatments are experiencing decreased potency due to severe host toxicity and the emergence of fungal drug resistance. As such, new targets and their corresponding synthetic pathways need to be explored for drug development purposes. In this context, galactofuranose residues, which are employed in fungal cell wall construction, but are notably absent in animals, represent an appealing target. Herein we present the structural and biochemical characterization of UDP-galactose-4-epimerase from Aspergillus nidulans which produces the precursor UDP-galactopyranose required for galactofuranose synthesis. Examination of the structural model revealed both NAD+ and UDP-glucopyranose were bound within the active site cleft in a near identical fashion to that found in the Human epimerase. Mutational studies on the conserved catalytic motif support a similar mechanism to that established for the Human counterpart is likely operational within the A. nidulans epimerase. While the Km and kcat for the enzyme were determined to be 0.11 mM and 12.8 s-1, respectively, a single point mutation, namely L320C, activated the enzyme towards larger N-acetylated substrates. Docking studies designed to probe active site affinity corroborate the experimentally determined activity profiles and support the kinetic inhibition results.
PMCID: PMC3792076  PMID: 24116166
14.  Structure modeling and comparative genomics for epimerase enzyme (Gal10p) 
Bioinformation  2010;5(6):266-270.
The Gal10p (UDP-Galactose 4-epimerase) protein is known for regulation of D-galactose metabolism. It catalyzes the inter-conversion between UDPgalactose and UDP-glucose. Knowledge of protein structure, neighboring interacting partners as well as functional residues of the Gal10p is crucial for carry out its function. These problems are still uncovered in case of the Epimerase enzyme. Structure of Epimerase enzyme has already been determined in S.cerevisiae and E.coli, however, no structural information for this protein is available for K.lactis. We used the homology modeling approach to model the structure of Gal10p in K.lactis. Furthermore, functional residues were predicted for modeled Gal10 protein and the strength of interaction between Gal10p and other Gal proteins was carried out by protein ‐protein interaction studies. The interaction studies revealed that the affinity of Gal10p for other Gal proteins vary in different organisms. Sequence and structure comparison of Epimerase enzyme showed that the orthologs in K.lactis and S.cervisiae are more similar to each other as compared to the ortholog in E.coli .The studies carried by us will help in better understanding of the galactose metabolism. The above studies may be applied to Human Gal10p, where it can help in gaining useful insight into Galactosemia disease.
PMCID: PMC3055706  PMID: 21364830
GAL protein; Gal10p; D-galactose metabolism; functional site prediction servers; RMSD;  protein-protein interaction
15.  P1 Trisaccharide (Galα1,4Galβ1,4GlcNAc) Synthesis by Enzyme Glycosylation Reactions Using Recombinant Escherichia coli 
The frequency of Escherichia coli infection has lead to concerns over pathogenic bacteria in our food supply and a demand for therapeutics. Glycolipids on gut cells serve as receptors for the Shiga-like toxin produced by E. coli. Oligosaccharide moiety analogues of these glycolipids can compete with receptors for the toxin, thus acting as antibacterials. An enzymatic synthesis of the P1 trisaccharide (Galα1,4Galβ1,4GlcNAc), one of the oligosaccharide analogues, was assessed in this study. In the proposed synthetic pathway, UDP-glucose was generated from sucrose with an Anabaena sp. sucrose synthase and then converted with an E. coli UDP-glucose 4-epimerase to UDP-galactose. Two molecules of galactose were linked to N-acetylglucosamine subsequently with a Helicobacter pylori β-l,4-galactosyltransferase and a Neisseria meningitidis α-1,4-galactosyltransferase to produce one molecule of P1 trisaccharide. The four enzymes were coexpressed in a single genetically engineered E. coli strain that was then permeabilized and used to catalyze the enzymatic reaction. P1 trisaccharide was accumulated up to 50 mM (5.4 g in a 200-ml reaction volume), with a 67% yield based on the consumption of N-acetylglucosamine. This study provides an efficient approach for the preparative-scale synthesis of P1 trisaccharide with recombinant bacteria.
PMCID: PMC154772  PMID: 12676690
16.  Structure and Mechanism of Human UDP-glucose 6-Dehydrogenase 
The Journal of Biological Chemistry  2011;286(27):23877-23887.
Elevated production of the matrix glycosaminoglycan hyaluronan is strongly implicated in epithelial tumor progression. Inhibition of synthesis of the hyaluronan precursor UDP-glucuronic acid (UDP-GlcUA) therefore presents an emerging target for cancer therapy. Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes, in two NAD+-dependent steps without release of intermediate aldehyde, the biosynthetic oxidation of UDP-glucose (UDP-Glc) to UDP-GlcUA. Here, we present a structural characterization of the hUGDH reaction coordinate using crystal structures of the apoenzyme and ternary complexes of the enzyme bound with UDP-Glc/NADH and UDP-GlcUA/NAD+. The quaternary structure of hUGDH is a disc-shaped trimer of homodimers whose subunits consist of two discrete α/β domains with the active site located in the interdomain cleft. Ternary complex formation is accompanied by rigid-body and restrained movement of the N-terminal NAD+ binding domain, sequestering substrate and coenzyme in their reactive positions through interdomain closure. By alternating between conformations in and out of the active site during domain motion, Tyr14, Glu161, and Glu165 participate in control of coenzyme binding and release during 2-fold oxidation. The proposed mechanism of hUGDH involves formation and breakdown of thiohemiacetal and thioester intermediates whereby Cys276 functions as the catalytic nucleophile. Stopped-flow kinetic data capture the essential deprotonation of Cys276 in the course of the first oxidation step, allowing the thiolate side chain to act as a trap of the incipient aldehyde. Because thiohemiacetal intermediate accumulates at steady state under physiological reaction conditions, hUGDH inhibition might best explore ligand binding to the NAD+ binding domain.
PMCID: PMC3129169  PMID: 21502315
Dehydrogenase; Enzyme Mechanisms; Enzyme Structure; Glycosaminoglycan; Hyaluronate; NAD; Tumor Marker; Conformational Changes and Catalysis; Covalent Intermediate; UDP-glucuronic Acid
17.  Adenosine Analogues as Selective Inhibitors of Glyceraldehyde-3-phosphate Dehydrogenase of Trypanosomatidae via Structure-Based Drug Design 
Journal of medicinal chemistry  2001;44(13):2080-2093.
In our continuation of the structure-based design of anti-trypanosomatid drugs, parasite-selective adenosine analogues were identified as low micromolar inhibitors of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Crystal structures of Trypanosoma brucei, Trypanosoma cruzi, Leishmania mexicana, and human GAPDH’s provided details of how the adenosyl moiety of NAD+ interacts with the proteins, and this facilitated the understanding of the relative affinities of a series of adenosine analogues for the various GAPDH’s. From exploration of modifications of the naphthalenemethyl and benzamide substituents of a lead compound, N6-(1-naphthalenemethyl)-2′-deoxy-2′-(3-methoxybenzamido)adenosine (6e), N6-(substituted-naphthalenemethyl)-2′-deoxy-2′-(substituted-benzamido)adenosine analogues were investigated. N6-(1-Naphthalenemethyl)-2′-deoxy-2′-(3,5-dimethoxybenzamido)adenosine (6m), N6-[1-(3-hydroxy-naphthalene)methyl]-2′-deoxy-2′-(3,5-dimethoxybenzamido)adenosine (7m), N6-[1-(3-methoxy-naphthalene)methyl]-2′-deoxy-2′-(3,5-dimethoxybenzamido)adenosine (9m), N6-(2-naphthalene-methyl)-2′-deoxy-2′-(3-methoxybenzamido)adenosine (11e), and N6-(2-naphthalenemethyl)-2′-deoxy-2′-(3,5-dimethoxybenzamido)adenosine (11m) demonstrated a 2- to 3-fold improvement over 6e and a 7100- to 25000-fold improvement over the adenosine template. IC50’s of these compounds were in the range 2–12 μM for T. brucei, T. cruzi, and L. mexicana GAPDH’s, and these compounds did not inhibit mammalian GAPDH when tested at their solubility limit. To explore more thoroughly the structure–activity relationships of this class of compounds, a library of 240 N6-(substituted)-2′-deoxy-2′-(amido)adenosine analogues was generated using parallel solution-phase synthesis with N6 and C2′ substituents chosen on the basis of computational docking scores. This resulted in the identification of 40 additional compounds that inhibit parasite GAPDH’s in the low micromolar range. We also explored adenosine analogues containing 5′-amido substituents and found that 2′,5′-dideoxy-2′-(3,5-dimethoxy-benzamido)-5′-(diphenylacetamido)adenosine (49) displays an IC50 of 60–100 μM against the three parasite GAPDH’s.
PMCID: PMC2957370  PMID: 11405646
18.  Expression, purification, crystallization and preliminary X-ray diffraction analysis of galactokinase from Pyrococcus horikoshii  
Galactokinase from P. horikoshii has been crystallized in both the apo form and as a ternary complex with α-d-galactose and an ATP analogue. The crystals were characterized by X-ray diffraction. The kinetic parameters of the enzyme were determined.
Galactokinase (EC catalyzes the ATP-dependent phosphorylation of α-­d-­galactose to α-d-galactose-1-phosphate, in an additional metabolic branch of glycolysis. The apo-form crystal structure of the enzyme has not yet been elucidated. Crystals of galactokinase from Pyrococcus horikoshii were prepared in both the apo form and as a ternary complex with α-d-galactose and an ATP analogue. Diffraction data sets were collected to 1.24 Å resolution for the apo form and to 1.7 Å for the ternary complex form using synchrotron radiation. The apo-form crystals belong to space group C2, with unit-cell parameters a = 108.08, b = 38.91, c = 81.57 Å, β = 109.8°. The ternary complex form was isomorphous with the apo form, except for the length of the a axis. The galactokinase activity of the enzyme was confirmed and the kinetic parameters at 323 K were determined.
PMCID: PMC2150956  PMID: 16511293
galactokinase; Leloir pathway; glycolysis; Pyrococcus horikoshii
19.  Crystallization and preliminary X-ray diffraction analysis of the putative aldose 1-epimerase YeaD from Escherichia coli  
The putative aldose 1-epimerase YeaD from Escherichia coli was crystallized and diffraction data were collected to a resolution of 1.9 Å.
Escherichia coli YeaD (ecYeaD) is suggested to be a member of the galactose mutarotase-like superfamily. Galactose mutarotase is an enzyme that converts α-galactose to β-galactose. The known structures of these galactose mutarotase-like proteins are similar to those of galactose mutarotases, with the catalytic residues being conserved, but there are some differences between them in the substrate-binding pocket. In order to reveal the specificity of ecYeaD, a three-dimensional structure is essential. Full-length ecYeaD with an additional 6×His tag at the C-­terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 4000 as a precipitant at 283 K. An X-ray diffraction data set was collected to a resolution of 1.9 Å from a single flash-cooled crystal that belonged to space group P212121.
PMCID: PMC2917301  PMID: 20693678
aldose 1-epimerases; YeaD; Escherichia coli; galactose mutarotases
20.  Characterization, Expression, and Mutation of the Lactococcus lactis galPMKTE Genes, Involved in Galactose Utilization via the Leloir Pathway 
Journal of Bacteriology  2003;185(3):870-878.
A cluster containing five similarly oriented genes involved in the metabolism of galactose via the Leloir pathway in Lactococcus lactis subsp. cremoris MG1363 was cloned and characterized. The order of the genes is galPMKTE, and these genes encode a galactose permease (GalP), an aldose 1-epimerase (GalM), a galactokinase (GalK), a hexose-1-phosphate uridylyltransferase (GalT), and a UDP-glucose 4-epimerase (GalE), respectively. This genetic organization reflects the order of the metabolic conversions during galactose utilization via the Leloir pathway. The functionality of the galP, galK, galT, and galE genes was shown by complementation studies performed with both Escherichia coli and L. lactis mutants. The GalP permease is a new member of the galactoside-pentose-hexuronide family of transporters. The capacity of GalP to transport galactose was demonstrated by using galP disruption mutant strains of L. lactis MG1363. A galK deletion was constructed by replacement recombination, and the mutant strain was not able to ferment galactose. Disruption of the galE gene resulted in a deficiency in cell separation along with the appearance of a long-chain phenotype when cells were grown on glucose as the sole carbon source. Recovery of the wild-type phenotype for the galE mutant was obtained either by genetic complementation or by addition of galactose to the growth medium.
PMCID: PMC142802  PMID: 12533462
21.  Identification of N-Acetylhexosamine 1-Kinase in the Complete Lacto-N-Biose I/Galacto-N-Biose Metabolic Pathway in Bifidobacterium longum▿  
Applied and Environmental Microbiology  2007;73(20):6444-6449.
We have determined the functions of the enzymes encoded by the lnpB, lnpC, and lnpD genes, located downstream of the lacto-N-biose phosphorylase gene (lnpA), in Bifidobacterium longum JCM1217. The lnpB gene encodes a novel kinase, N-acetylhexosamine 1-kinase, which produces N-acetylhexosamine 1-phosphate; the lnpC gene encodes UDP-glucose hexose 1-phosphate uridylyltransferase, which is also active on N-acetylhexosamine 1-phosphate; and the lnpD gene encodes a UDP-glucose 4-epimerase, which is active on both UDP-galactose and UDP-N-acetylgalactosamine. These results suggest that the gene operon lnpABCD encodes a previously undescribed lacto-N-biose I/galacto-N-biose metabolic pathway that is involved in the intestinal colonization of bifidobacteria and that utilizes lacto-N-biose I from human milk oligosaccharides or galacto-N-biose from mucin sugars.
PMCID: PMC2075035  PMID: 17720833
22.  The nucleotide-binding site of Aquifex aeolicus LpxC 
The structure of UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) in complex with UDP is reported. The complex allows for a description of how the enzyme recognizes and binds a nucleotide moiety and enables the construction of an LpxC-substrate model.
The structure of recombinant Aquifex aeolicus UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) in complex with UDP has been determined to a resolution of 2.2 Å. Previous studies have characterized the binding sites of the fatty-acid and sugar moieties of the substrate, UDP-(3-O-hydroxymyristoyl)-N-­acetylglucosamine, but not that of the nucleotide. The uracil-binding site is constructed from amino acids that are highly conserved across species. Hydrophobic associations with the Phe155 and Arg250 side chains in combination with hydrogen-bonding interactions with the main chain of Glu154 and the side chains of Tyr151 and Lys227 position the base. The phosphate and ribose groups are directed away from the active site and interact with Arg137, Lys156, Glu186 and Arg250. The orientation of the phosphate-ribose tail is not conducive to catalysis, perhaps owing to the position of an inhibitory Zn2+. However, based on the position of uracil revealed in this study and on the previously reported complex of LpxC with an inhibitor, a model is proposed for substrate binding.
PMCID: PMC2225228  PMID: 17077484
lipid A; Aquifex aeolicus; LpxC
23.  Substrate Channel Flexibility in Pseudomonas aeruginosa MurB Accommodates Two Distinct Substrates 
PLoS ONE  2013;8(6):e66936.
Biosynthesis of UDP-N-acetylmuramic acid in bacteria is a committed step towards peptidoglycan production. In an NADPH- and FAD-dependent reaction, the UDP-N-acetylglucosamine-enolpyruvate reductase (MurB) reduces UDP-N-acetylglucosamine-enolpyruvate to UDP-N-acetylmuramic acid. We determined the three-dimensional structures of the ternary complex of Pseudomonas aeruginosa MurB with FAD and NADP+ in two crystal forms to resolutions of 2.2 and 2.1 Å, respectively, to investigate the structural basis of the first half-reaction, hydride transfer from NADPH to FAD. The nicotinamide ring of NADP+ stacks against the si face of the isoalloxazine ring of FAD, suggesting an unusual mode of hydride transfer to flavin. Comparison with the structure of the Escherichia coli MurB complex with UDP-N-acetylglucosamine-enolpyruvate shows that both substrates share the binding site located between two lobes of the substrate-binding domain III, consistent with a ping pong mechanism with sequential substrate binding. The nicotinamide and the enolpyruvyl moieties are strikingly well-aligned upon superimposition, both positioned for hydride transfer to and from FAD. However, flexibility of the substrate channel allows the non-reactive parts of the two substrates to bind in different conformations. A potassium ion in the active site may assist in substrate orientation and binding. These structural models should help in structure-aided drug design against MurB, which is essential for cell wall biogenesis and hence bacterial survival.
PMCID: PMC3689657  PMID: 23805286
24.  Biosynthesis of Galactofuranose in Kinetoplastids: Novel Therapeutic Targets for Treating Leishmaniasis and Chagas' Disease 
Enzyme Research  2011;2011:415976.
Cell surface proteins of parasites play a role in pathogenesis by modulating mammalian cell recognition and cell adhesion during infection. β-Galactofuranose (Galf) is an important component of glycoproteins and glycolipids found on the cell surface of Leishmania spp. and Trypanosoma cruzi. β-Galf-containing glycans have been shown to be important in parasite-cell interaction and protection against oxidative stress. Here, we discuss the role of β-Galf in pathogenesis and recent studies on the Galf-biosynthetic enzymes: UDP-galactose 4′ epimerase (GalE), UDP-galactopyranose mutase (UGM), and UDP-galactofuranosyl transferase (GalfT). The central role in Galf formation, its unique chemical mechanism, and the absence of a homologous enzyme in humans identify UGM as the most attractive drug target in the β-Galf-biosynthetic pathway in protozoan parasites.
PMCID: PMC3112513  PMID: 21687654
25.  Structural Basis of Cooperativity in Human UDP-Glucose Dehydrogenase 
PLoS ONE  2011;6(10):e25226.
UDP-glucose dehydrogenase (UGDH) is the sole enzyme that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. The product is used in xenobiotic glucuronidation in hepatocytes and in the production of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers, while inhibition of UGDH diminished tumor angiogenesis in vivo. A better understanding of the conformational changes occurring during the UGDH reaction cycle will pave the way for inhibitor design and potential cancer therapeutics.
Previously, the substrate-bound of UGDH was determined to be a symmetrical hexamer and this regular symmetry is disrupted on binding the inhibitor, UDP-α-D-xylose. Here, we have solved an alternate crystal structure of human UGDH (hUGDH) in complex with UDP-glucose at 2.8 Å resolution. Surprisingly, the quaternary structure of this substrate-bound protein complex consists of the open homohexamer that was previously observed for inhibitor-bound hUGDH, indicating that this conformation is relevant for deciphering elements of the normal reaction cycle.
In all subunits of the present open structure, Thr131 has translocated into the active site occupying the volume vacated by the absent active water and partially disordered NAD+ molecule. This conformation suggests a mechanism by which the enzyme may exchange NADH for NAD+ and repolarize the catalytic water bound to Asp280 while protecting the reaction intermediates. The structure also indicates how the subunits may communicate with each other through two reaction state sensors in this highly cooperative enzyme.
PMCID: PMC3184952  PMID: 21984906

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