Galactofuranose (Galf) residues are present in the cell wall glycoconjugates of numerous pathogenic microbes. UDP-galactofuranose (UDP-Galf), the biosynthetic precursor of Galf-containing glycoconjugates, is produced from UDP-galactopyranose (UDP-Galp) by the flavoenzyme UDP-galactopyranose mutase (UGM). The gene encoding UGM (glf) is essential for the viability of pathogens, including Mycobacterium tuberculosis, and this finding underscores the need to understand how UGM functions. Considerable effort has been devoted to elucidating the catalytic mechanism of UGM, but progress has been hindered by a lack of structural data for an enzyme-substrate complex. Such data could reveal not only the substrate binding interactions but how UGM can act preferentially on two very different substrates, UDP-Galp and UDP-Galf, yet avoid other structurally related UDP-sugars present in the cell. Herein, we describe the first structure of a UGM-ligand complex, which provides insight into the catalytic mechanism and the molecular basis for substrate selectivity. The structure of UGM from Klebsiella pneumoniae bound to the substrate analog UDP-glucose (UDP-Glc) was solved by X-ray crystallographic methods and refined to 2.5 Å resolution. The ligand is proximal to the cofactor, a finding that is consistent with a proposed mechanism in which the reduced flavin engages in covalent catalysis. Despite this proximity, the glucose ring of the substrate analog is positioned such that it disfavors covalent catalysis. This orientation is consistent with data indicating that UDP-Glc is not a substrate for UGM. The relative binding orientations of UDP-Galp and UDP-Glc were compared using saturation transfer difference-NMR. The results indicate that the uridine moiety occupies a similar location in both ligand complexes, and this relevant binding mode is defined by our structural data. In contrast, the orientations of the glucose and galactose sugar moieties differ. To understand the consequences of these differences, we derived a model for the productive UGM-substrate complex that highlights interactions that can contribute to catalysis and substrate discrimination.
UDP-galactopyranose mutase; Glf; cell wall biosynthesis; mycobacteria; galactofuranose
UDP-galactose 4′-epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose, an important step in galactose catabolism. Type III galactosemia, an inherited metabolic disease, is associated with mutations in human GALE. The V94M mutation has been associated with a very severe form of type III galactosemia. While a variety of structural and biochemical studies have been reported that elucidate differences between the wildtype and this mutant form of human GALE, little is known about the dynamics of the protein and how mutations influence structure and function. We performed molecular dynamics simulations on the wildtype and V94M enzyme in different states of substrate and cofactor binding. In the mutant, the average distance between the substrate and both a key catalytic residue (Tyr-157) and the enzyme-bound NAD+ cofactor and the active site dynamics are altered making substrate binding slightly less stable. However, overall stability or dynamics of the protein is not altered. This is consistent with experimental findings that the impact is largely on the turnover number (kcat), with less substantial effects on Km. Active site fluctuations were found to be correlated in enzyme with substrate bound to just one of the subunits in the homodimer suggesting inter-subunit communication. Greater active site loop mobility in human GALE compared to the equivalent loop in Escherichia coli GALE explains why the former can catalyze the interconversion of UDP-N-acetylgalactosemine and UDP-N-acetylglucosamine while the bacterial enzyme cannot. This work illuminates molecular mechanisms of disease and may inform the design of small molecule therapies for type III galactosemia.
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.
kinetoplastids; sugar nucleotide metabolism; Trypanosoma brucei; UDP-glucose; UDP-glucose pyrophosphorylase
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.
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.
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.
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.
3β-hydroxysteroid dehydrogenase; short-chain oxidoreductase; 3D model structure; bioinformatics; rational proteomics; structure based mutagenesis; structure-function relationship
Background: UDP-galactopyranose mutase (UGM) is a critical enzyme for the proper formation of the cell wall of pathogenic microbes.
Results: The structure of UGM in complex with substrate reveals novel features of substrate binding.
Conclusion: Oxidation and reduction of the flavin cofactor causes rearrangements in the active site that affect substrate binding.
Significance: These are the first structures of UGM from a eukaryotic pathogen.
UDP-galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyzes the reversible conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). As in prokaryotic UGMs, the flavin needs to be reduced for the enzyme to be active. Here we present the first eukaryotic UGM structures from Aspergillus fumigatus (AfUGM). The structures are of UGM alone, with the substrate UDP-Galp and with the inhibitor UDP. Additionally, we report the structures of AfUGM bound to substrate with oxidized and reduced flavin. These structures provide insight into substrate recognition and structural changes observed upon substrate binding involving the mobile loops and the critical arginine residues Arg-182 and Arg-327. Comparison with prokaryotic UGM reveals that despite low sequence identity with known prokaryotic UGMs the overall fold is largely conserved. Structural differences between prokaryotic UGM and AfUGM result from inserts in AfUGM. A notable difference from prokaryotic UGMs is that AfUGM contains a third flexible loop (loop III) above the si-face of the isoalloxazine ring that changes position depending on the redox state of the flavin cofactor. This loop flipping has not been observed in prokaryotic UGMs. In addition we have determined the crystals structures and steady-state kinetic constants of the reaction catalyzed by mutants R182K, R327K, R182A, and R327A. These results support our hypothesis that Arg-182 and Arg-327 play important roles in stabilizing the position of the diphosphates of the nucleotide sugar and help to facilitate the positioning of the galactose moiety for catalysis.
Carbohydrate-binding Protein; Cell Wall; Enzyme Mutation; Enzyme Structure; Membrane Biogenesis
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.
epimerases; galactose metabolism; short-chain dehydrogenases; Leloir pathway
Deficiency of UDP-galactose 4′-epimerase is implicated in type III galactosemia. Two variants, p.K161N-hGALE and p.D175N-hGALE, have been previously found in combination with other alleles in patients with a mild form of the disease. Both variants were studied in vivo and in vitro and showed different levels of impairment. p.K161N-hGALE was severely impaired with substantially reduced enzymatic activity, increased thermal stability, reduced cofactor binding and inability to rescue the galactose-sensitivity of gal10-null yeast. Interestingly p.K161N-hGALE showed less impairment of activity with UDP-N-acetylgalactosamine in comparison to UDP-galactose. Differential scanning fluorimetry revealed that p.K161N-hGALE was more stable than the wild-type protein and only changed stability in the presence of UDP-N-acetylglucosamine and NAD+. p.D175N-hGALE essentially rescued the galactose-sensitivity of gal10-null yeast, was less stable than the wild-type protein but showed increased stability in the presence of substrates and cofactor. We postulate that p.K161N-hGALE causes its effects by abolishing an important interaction between the protein and the cofactor, whereas p.D175N-hGALE is predicted to remove a stabilizing salt bridge between the ends of two α-helices that contain residues that interact with NAD+. These results suggest that the cofactor binding is dynamic and that its loss results in significant structural changes that may be important in disease causation.
Type III galactosemia; yeast model; GALE; disease-associated mutation; UDP-galactose 4′-epimerase; Differential scanning fluorimetry
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.
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.
GalE, UDP-galactose-4′-epimerase; UDP-GlcA, UDP-glucuronic acid; UXS, UDP-xylose synthase; UDP-xylose; UDP-galactose; Trichomonas vaginalis
Human African trypanosomiasis (HAT), a parasitic protozoal disease, is caused primarily by two subspecies of Trypanosoma brucei. HAT is a re-emerging disease and currently threatens millions of people in sub-Saharan Africa. Many affected people live in remote areas with limited access to health services and, therefore, rely on traditional herbal medicines for treatment.
A molecular docking study has been carried out on phytochemical agents that have been previously isolated and characterized from Nigerian medicinal plants, either known to be used ethnopharmacologically to treat parasitic infections or known to have in-vitro antitrypanosomal activity. A total of 386 compounds from 19 species of medicinal plants were investigated using in-silico molecular docking with validated Trypanosoma brucei protein targets that were available from the Protein Data Bank (PDB): Adenosine kinase (TbAK), pteridine reductase 1 (TbPTR1), dihydrofolate reductase (TbDHFR), trypanothione reductase (TbTR), cathepsin B (TbCatB), heat shock protein 90 (TbHSP90), sterol 14α-demethylase (TbCYP51), nucleoside hydrolase (TbNH), triose phosphate isomerase (TbTIM), nucleoside 2-deoxyribosyltransferase (TbNDRT), UDP-galactose 4′ epimerase (TbUDPGE), and ornithine decarboxylase (TbODC).
This study revealed that triterpenoid and steroid ligands were largely selective for sterol 14α-demethylase; anthraquinones, xanthones, and berberine alkaloids docked strongly to pteridine reductase 1 (TbPTR1); chromenes, pyrazole and pyridine alkaloids preferred docking to triose phosphate isomerase (TbTIM); and numerous indole alkaloids showed notable docking energies with UDP-galactose 4′ epimerase (TbUDPGE). Polyphenolic compounds such as flavonoid gallates or flavonoid glycosides tended to be promiscuous docking agents, giving strong docking energies with most proteins.
This in-silico molecular docking study has identified potential biomolecular targets of phytochemical components of antitrypanosomal plants and has determined which phytochemical classes and structural manifolds likely target trypanosomal enzymes. The results could provide the framework for synthetic modification of bioactive phytochemicals, de novo synthesis of structural motifs, and lead to further phytochemical investigations.
Traditional herbal medicine continues to play a key role in health, particularly in remote areas with limited access to “modern medicines”. Many plants are used in traditional Nigerian medicine to treat parasitic diseases. While many of these plants have shown notable activity against parasitic protozoa, in most cases the mode of activity is not known. That is, it is not known what biochemical entities are being targeted by the plant chemical constituents. In this work, we have carried out molecular docking studies of known phytochemicals from Nigerian medicinal plants used to treat human African trypanosomiasis (sleeping sickness) with known biochemical targets in the Trypanosoma brucei parasite. The goals of this study were to identify the protein targets that the medicinal plants are affecting and to discern general trends in protein target selectivity for phytochemical classes. In doing so, we have theoretically identified strongly interacting plant chemicals and their biomolecular targets. These results should lead to further research to verify the efficacy of the phytochemical agents as well as delineate possible modifications of the active compounds to increase potency or selectivity.
Background: Human UDP-xylose synthase (hUXS1) is responsible for conversion of UDP-glucuronic acid to UDP-xylose.
Results: Crystal structure, molecular dynamics simulations, and reaction course analysis give conclusive insight into the enzymatic mechanism in three catalytic steps.
Conclusion: Distortion of sugar pyranose ring in bound substrate facilitates enzymatic reaction.
Significance: A detailed mechanism for catalysis by hUXS1 is proposed.
UDP-xylose synthase (UXS) catalyzes decarboxylation of UDP-d-glucuronic acid to UDP-xylose. In mammals, UDP-xylose serves to initiate glycosaminoglycan synthesis on the protein core of extracellular matrix proteoglycans. Lack of UXS activity leads to a defective extracellular matrix, resulting in strong interference with cell signaling pathways. We present comprehensive structural and mechanistic characterization of the human form of UXS. The 1.26-Å crystal structure of the enzyme bound with NAD+ and UDP reveals a homodimeric short-chain dehydrogenase/reductase (SDR), belonging to the NDP-sugar epimerases/dehydratases subclass. We show that enzymatic reaction proceeds in three chemical steps via UDP-4-keto-d-glucuronic acid and UDP-4-keto-pentose intermediates. Molecular dynamics simulations reveal that the d-glucuronyl ring accommodated by UXS features a marked 4C1
chair to BO,3
boat distortion that facilitates catalysis in two different ways. It promotes oxidation at C4 (step 1) by aligning the enzymatic base Tyr147 with the reactive substrate hydroxyl and it brings the carboxylate group at C5 into an almost fully axial position, ideal for decarboxylation of UDP-4-keto-d-glucuronic acid in the second chemical step. The protonated side chain of Tyr147 stabilizes the enolate of decarboxylated C4 keto species (2H1
half-chair) that is then protonated from the Si face at C5, involving water coordinated by Glu120. Arg277, which is positioned by a salt-link interaction with Glu120, closes up the catalytic site and prevents release of the UDP-4-keto-pentose and NADH intermediates. Hydrogenation of the C4 keto group by NADH, assisted by Tyr147 as catalytic proton donor, yields UDP-xylose adopting the relaxed 4C1
chair conformation (step 3).
Carbohydrate Biosynthesis; Decarboxylase; Dehydrogenase; Enzyme Mechanisms; Enzyme Structure; Glycosaminoglycan; Enzyme Reaction Coordinate; Short-chain Dehydrogenase/Reductase; Substrate Distortion/Destabilization; UDP-glucuronic Acid
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.
Dehydrogenase; Enzyme Mechanisms; Enzyme Structure; Glycosaminoglycan; Hyaluronate; NAD; Tumor Marker; Conformational Changes and Catalysis; Covalent Intermediate; UDP-glucuronic Acid
In Escherichia coli and Salmonella enterica, the core oligosaccharide backbone of the lipopolysaccharide is modified by phosphoryl groups. The negative charges provided by these residues are important in maintaining the barrier function of the outer membrane. In contrast, Klebsiella pneumoniae lacks phosphoryl groups in its core oligosaccharide but instead contains galacturonic acid residues that are proposed to serve a similar function in outer membrane stability. GlaKP is a UDP-galacturonic acid C4-epimerase that provides UDP-galacturonic acid for core synthesis, and the enzyme was biochemically characterized because of its potentially important role in outer membrane stability. High-performance anion-exchange chromatography was used to demonstrate the UDP-galacturonic acid C4-epimerase activity of GlaKP, and capillary electrophoresis was used for activity assays. The reaction equilibrium favors UDP-galacturonic acid over UDP-glucuronic acid in a ratio of 1.4:1, with the Km for UDP-glucuronic acid of 13.0 μM. GlaKP exists as a dimer in its native form. NAD+/NADH is tightly bound by the enzyme and addition of supplementary NAD+ is not required for activity of the purified enzyme. Divalent cations have an unexpected inhibitory effect on enzyme activity. GlaKP was found to have a broad substrate specificity in vitro; it is capable of interconverting UDP-glucose/UDP-galactose and UDP-N-acetylglucosamine/UDP-N-acetylgalactosamine, albeit at much lower activity. The epimerase GalE interconverts UDP-glucose/UDP-galactose. Multicopy plasmid-encoded glaKP partially complemented a galE mutation in S. enterica and in K. pneumoniae; however, chromosomal glaKP could not substitute for galE in a K. pneumoniae galE mutant in vivo.
The anthocyanidin 3-O-glucosyltransferase from Clitoria ternatea (Ct3GT-A) was expressed in Escherichia coli, and the three-dimensional structure of Ct3GT-A was determined using X-ray crystallography. This report describes the architecture of Ct3GT-A, including the structures of the donor- and acceptor-binding sites.
Flowers of the butterfly pea (Clitoria ternatea) accumulate a group of polyacylated anthocyanins, named ternatins, in their petals. The first step in ternatin biosynthesis is the transfer of glucose from UDP-glucose to anthocyanidins such as delphinidin, a reaction catalyzed in C. ternatea by UDP-glucose:anthocyanidin 3-O-glucosyltransferase (Ct3GT-A; AB185904). To elucidate the structure–function relationship of Ct3GT-A, recombinant Ct3GT-A was expressed in Escherichia coli and its tertiary structure was determined to 1.85 Å resolution by using X-ray crystallography. The structure of Ct3GT-A shows a common folding topology, the GT-B fold, comprised of two Rossmann-like β/α/β domains and a cleft located between the N- and C-domains containing two cavities that are used as binding sites for the donor (UDP-Glc) and acceptor substrates. By comparing the structure of Ct3GT-A with that of the flavonoid glycosyltransferase VvGT1 from red grape (Vitis vinifera) in complex with UDP-2-deoxy-2-fluoro glucose and kaempferol, locations of the catalytic His-Asp dyad and the residues involved in recognizing UDP-2-deoxy-2-fluoro glucose were essentially identical in Ct3GT-A, but certain residues of VvGT1 involved in binding kaempferol were found to be substituted in Ct3GT-A. These findings are important for understanding the differentiation of acceptor-substrate recognition in these two enzymes.
glucosylation; glucosyltransferase; anthocyanidin; crystal structure
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.
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.
Lipopolysaccharide; N-acetylglucosamine; Rossmann fold; 4-Epimerase; Galactose metabolism
2,3-diacetamido-2,3-dideoxy-D-mannuronic acid or ManNAc3NAcA is an unusual dideoxysugar first identified nearly 30 years ago in the lipopolysaccharide of Pseudomonas aeruginosa O:3a,d. It has since been observed in other organisms including Bordetella pertussis, the causative agent of whooping cough. Five enzymes are required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetyl-D-glucosamine. Here we describe a structural study of WlbA, the NAD-dependent dehydrogenase that catalyzes the second step in the pathway, namely the oxidation of the C-3′ hydroxyl group on the UDP-linked sugar to a keto moiety and the reduction of NAD+ to NADH. This enzyme has been shown to use α-ketoglutarate as an oxidant to regenerate the reduced dinucleotide. For this investigation three different crystal structures were determined: the enzyme with bound NAD(H), the enzyme complexed with NAD(H) and α-ketoglutarate, and the enzyme complexed with NAD(H) and its substrate (UDP-N-acetyl-D-glucosaminuronic acid). The tetrameric enzyme assumes an unusual quaternary structure with the dinucleotides positioned quite closely to one another. Both α-ketoglutarate and the UDP-linked sugar bind in the WlbA active site with their carbon atoms (C-2 or C3′, respectively) abutting the re face of the cofactor. They are positioned at ~3 Å from the nicotinamide C-4. The UDP-linked sugar substrate adopts a high unusual curved conformation when bound in the WlbA active site cleft. Lys 101 and His 185 most likely play key roles in catalysis.
UDP-N-acetylmuramic acid:l-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan. The crystal structures of Haemophilus influenzae MurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg2+ and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-l-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn2+ have been determined to 1.85- and 1.7-Å resolution, respectively. These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine. An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound. The MurC active site clearly shows that the γ-phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group. These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates.
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.
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.
African sleeping sickness; molecular dynamics; protein structure; TbGalE; Trypanosoma brucei; UDP-Galactose-4′-Epimerase
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.
TbGalE; Trypanosoma brucei; UDP-Galactose-4’-Epimerase; African Sleeping Sickness; Molecular Dynamics; Protein Structure
The cell wall polysaccharide of Streptococcus gordonii 38 functions as a coaggregation receptor for surface adhesins on other members of the oral biofilm community. The structure of this receptor polysaccharide (RPS) is defined by a heptasaccharide repeat that includes a GalNAcβ1→3Gal-containing recognition motif. The same RPS has now been identified from S. gordonii AT, a partially sequenced strain. PCR primers designed from sequences in the genomic database of strain AT were used to identify and partially characterize the S. gordonii 38 RPS gene cluster. This cluster includes genes for seven putative glycosyltransferases, a polysaccharide polymerase (Wzy), an oligosaccharide repeating unit transporter (Wzx), and a galactofuranose mutase, the enzyme that promotes synthesis of UDP-Galf, one of five predicted RPS precursors. Genes outside this region were identified for the other four nucleotide-linked sugar precursors of RPS biosynthesis, namely, those for formation of UDP-Glc, UDP-Gal, UDP-GalNAc, and dTDP-Rha. Two genes for putative galactose 4-epimerases were identified. The first, designated galE1, was identified as a pseudogene in the galactose operon, and the second, designated galE2, was transcribed with three of the four genes for dTDP-Rha biosynthesis (i.e., rmlA, rmlC, and rmlB). Insertional inactivation of galE2 abolished (i) RPS production, (ii) growth on galactose, and (iii) both UDP-Gal and UDP-GalNAc 4-epimerase activities in cell extracts. Repair of the galE1 pseudogene in this galE2 mutant restored growth on galactose but not RPS production. Cell extracts containing functional GalE1 but not GalE2 contained UDP-Gal 4-epimerase but not UDP-GalNAc 4-epimerase activity. Thus, provision of both UDP-Gal and UDP-GalNAc for RPS production by S. gordonii 38 depends on the dual specificity of the epimerase encoded by galE2.
Cationic Antimicrobial Peptides (CAMPs)
represent a first line
of defense against bacterial colonization. When fighting Gram-negative
bacteria, CAMPs initially interact electrostatically with the negatively
charged phosphate groups in lipid A and are thought to kill bacteria
by disrupting their membrane integrity. However, many human pathogens,
including Salmonella and Pseudomonas, have evolved lipid A modification mechanisms
that result in resistance to CAMPs and related antibiotics such as
Colistin. The addition of 4-amino-4-deoxy-l-Arabinose (Ara4N)
to a phosphate group in lipid A is one such modification, frequently
found in Pseudomonas isolated from
cystic fibrosis patients. The pathway for biosynthesis of Ara4N-lipid
A requires conversion of UDP-Glucuronic acid into UDP-Ara4N and subsequent
transfer of the amino-sugar to lipid A. ArnB is a pyridoxal-phosphate
(PLP) dependent transaminase that catalyzes a crucial step in the
pathway: synthesis of UDP-Ara4N from UDP-4-keto-pentose. Here we present
the 2.3 Å resolution crystal structure of an active site mutant
of ArnB (K188A) in complex with the reaction intermediate aldimine
formed by UDP-Ara4N and PLP. The sugar–nucleotide binding site
is in a cleft between the subunits of the ArnB dimer with the uracil
buried at the interface and the UDP ribose and phosphate groups exposed
to the solvent. The Ara4N moiety is found in the 4C1 conformation and its positioning, stabilized by interactions
with both the protein and cofactor, is compatible with catalysis.
The structure suggests strategies for the development of specific
inhibitors that may prove useful in the treatment of resistant bacteria
such as Pseudomonas found in cystic