Epimerase Enzyme Research Articles

Gene expression profile of Campylobacter jejuni in response to macrolide antibiotics.

Campylobacter jejuni is a foodborne pathogen that causes gastroenteritis in humans and has developed resistance to various antibiotics. The primary objective of this research was to examine the network of antibiotic resistance in C. jejuni. The study involved the wild and antibiotic-resistant strains placed in the presence and absence of antibiotics to review their gene expression profiles in response to ciprofloxacin via microarray. Differentially expressed genes (DEGs) analysis and Protein-Protein Interaction (PPI) Network studies were performed for these genes. The results showed that the resistance network of C. jejuni is modular, with different genes involved in bacterial motility, capsule synthesis, efflux, and amino acid and sugar synthesis. Antibiotic treatment resulted in the down-regulation of cluster genes related to translation, flagellum formation, and chemotaxis. In contrast, cluster genes involved in homeostasis, capsule formation, and cation efflux were up-regulated. The study also found that macrolide antibiotics inhibit the progression of C. jejuni infection by inactivating topoisomerase enzymes and increasing the activity of epimerase enzymes, trying to compensate for the effect of DNA twisting. Then, the bacterium limits the movement to conserve energy. Identifying the antibiotic resistance network in C. jejuni can aid in developing drugs to combat these bacteria. Genes involved in cell division, capsule formation, and substance transport may be potential targets for inhibitory drugs. Future research must be directed toward comprehending the underlying mechanisms contributing to the modularity of antibiotic resistance and developing strategies to disrupt and mitigate the growing threat of antibiotic resistance effectively.

Physiology and Pathophysiology of Heparan Sulfate in Animal Models: Its Biosynthesis and Degradation.

Heparan sulfate (HS) is a type of glycosaminoglycan that plays a key role in a variety of biological functions in neurology, skeletal development, immunology, and tumor metastasis. Biosynthesis of HS is initiated by a link of xylose to Ser residue of HS proteoglycans, followed by the formation of a linker tetrasaccharide. Then, an extension reaction of HS disaccharide occurs through polymerization of many repetitive units consisting of iduronic acid and N-acetylglucosamine. Subsequently, several modification reactions take place to complete the maturation of HS. The sulfation positions of N-, 2-O-, 6-O-, and 3-O- are all mediated by specific enzymes that may have multiple isozymes. C5-epimerization is facilitated by the epimerase enzyme that converts glucuronic acid to iduronic acid. Once these enzymatic reactions have been completed, the desulfation reaction further modifies HS. Apart from HS biosynthesis, the degradation of HS is largely mediated by the lysosome, an intracellular organelle with acidic pH. Mucopolysaccharidosis is a genetic disorder characterized by an accumulation of glycosaminoglycans in the body associated with neuronal, skeletal, and visceral disorders. Genetically modified animal models have significantly contributed to the understanding of the in vivo role of these enzymes. Their role and potential link to diseases are also discussed.

Molecular dynamics, residue network analysis, and cross-correlation matrix to characterize the deleterious missense mutations in GALE causing galactosemia III.

Epimerase-deficiency galactosemia (EDG) is caused by mutations in the UDP-galactose 4'-epimerase enzyme, encoded by gene GALE. Catalyzing the last reaction in the Leloir pathway, UDP-galactose-4-epimerase catalyzes the interconversion of UDP-galactose and UDP-glucose. This study aimed to use in-depth computational strategies to prioritize the pathogenic missense mutations in GALE protein and investigate the systemic behavior, conformational spaces, atomic motions, and cross-correlation matrix of the GALE protein. We searched four databases (dbSNP, ClinVar, UniProt, and HGMD) and major biological literature databases (PubMed, Science Direct, and Google Scholar), for missense mutations that are associated with EDG patients, our search yielded 190 missense mutations. We applied a systematic computational prediction pipeline, including pathogenicity, stability, biochemical, conservational, protein residue contacts, and structural analysis, to predict the pathogenicity of these mutations. We found three mutations (p.K161N, p.R239W, and p.G302D) with a severe phenotype in patients with EDG that correlated with our computational prediction analysis; thus, they were selected for further structural and simulation analyses to compute the flexibility and stability of the mutant GALE proteins. The three mutants were subjected to molecular dynamics simulation (MDS) with native protein for 200 ns using GROMACS. The MDS demonstrated that these mutations affected the beta-sheets and helical region that are responsible for the catalytic activity; subsequently, affects the stability and flexibility of the mutant proteins along with a decrease and more deviations in compactness when compared to that of a native. Also, three mutations created major variations in the combined atomic motions of the catalytic and C-terminal regions. The network analysis of the residues in the native and three mutant protein structures showed disturbed residue contacts occurred owing to the missense mutations. Our findings help to understand the structural behavior of a protein owing to mutation and are intended to serve as a platform for prioritizing mutations, which could be potential targets for drug discovery and development of targeted therapeutics.

Altered Ovarian Inositol Ratios May Account for Pathological Steroidogenesis in PCOS.

The presence of abnormal ovarian ratios of myo-inositol (MI) to D-chiro-inositol (DCI) is a recurrent feature in PCOS. Available evidence suggests that MI and DCI may modulate steroid biosynthesis, likely in an opposite manner. Specifically, MI seems to induce estrogen production, while DCI has a role in the synthesis of androgens. Elevated insulin levels, generally associated with PCOS, alter the physiological MI/DCI ratio, increasing MI-to-DCI conversion through activation of a specific epimerase enzyme. DCI directly increases testosterone biosynthesis in thecal cells and reduces its conversion to estradiol by downregulating aromatase enzyme in granulosa cells. This manuscript reviews the literature that supports the connection between altered MI/DCI ratios and pathological steroidogenesis observed in PCOS women. Furthermore, it discusses the application of inositol-based treatment protocols in managing PCOS symptoms and improving the quality of patients’ life.

Bacterial synthesis of D-amino acids.

Recent work has shed light on the abundance and diversity of D-amino acids in bacterial extracellular/periplasmic molecules, bacterial cell culture, and bacteria-rich environments. Within the extracellular/periplasmic space, D-amino acids are necessary components of peptidoglycan, and disruption of their synthesis leads to cell death. As such, enzymes responsible for D-amino acid synthesis are promising targets for antibacterial compounds. Further, bacteria are shown to incorporate a diverse collection of D-amino acids into their peptidoglycan, and differences in D-amino acid incorporation may occur in response to differences in growth conditions. Certain D-amino acids can accumulate to millimolar levels in cell culture, and their synthesis is proposed to foretell movement from exponential growth phase into stationary phase. While enzymes responsible for synthesis of D-amino acids necessary for peptidoglycan (D-alanine and D-glutamate) have been characterized from a number of different bacteria, the D-amino acid synthesis enzymes characterized to date cannot account for the diversity of D-amino acids identified in bacteria or bacteria-rich environments. Free D-amino acids are synthesized by racemization or epimerization at the α-carbon of the corresponding L-amino acid by amino acid racemase or amino acid epimerase enzymes. Additionally, D-amino acids can be synthesized by stereospecific amination of α-ketoacids. Below, we review the roles of D-amino acids in bacterial physiology and biotechnology, and we describe the known mechanisms by which they are synthesized by bacteria.

A new robust kinetic assay for DAP epimerase activity

DAP epimerase is the penultimate enzyme in the lysine biosynthesis pathway. The most versatile assay for DAP epimerase catalytic activity employs a coupled DAP epimerase-DAP dehydrogenase enzyme system with a commercial mixture of DAP isomers as substrate. DAP dehydrogenase converts meso-DAP to THDP with concomitant reduction of NADP(+) to NADPH. We show that at high concentrations, accumulation of NADPH results in inhibition of DAPDH, resulting in spurious kinetic data. A new assay has been developed employing DAP decarboxylase that allows the reliable characterisation of DAP epimerase enzyme kinetics.

Structure modeling and comparative genomics for epimerase enzyme (Gal10p).

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.

D-Cysteine occurrence in thiostrepton may not necessitate an epimerase

The natural product thiopeptide antibiotic thiostrepton is shown to undergo facile epimerization at its thiazoline residue in favor of the naturally observed D-configuration, suggesting that a classical epimerase enzyme may not be involved in its biosynthesis.

Biosynthesis of a Rare Di-N-Acetylated Sugar in the Lipopolysaccharides of both Pseudomonas aeruginosa and Bordetella pertussis Occurs via an Identical Scheme despite Different Gene Clusters

Pseudomonas aeruginosa and Bordetella pertussis produce lipopolysaccharide (LPS) that contains 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid (D-ManNAc3NAcA). A five-enzyme biosynthetic pathway that requires WbpA, WbpB, WbpE, WbpD, and WbpI has been proposed for the production of this sugar in P. aeruginosa, based on analysis of genes present in the B-band LPS biosynthesis cluster. In the analogous B. pertussis cluster, homologs of wbpB to wbpI were present, but a putative dehydrogenase gene was missing; therefore, the biosynthetic mechanism for UDP-D-ManNAc3NAcA was unclear. Nonpolar knockout mutants of each P. aeruginosa gene were constructed. Complementation analysis of the mutants demonstrated that B-band LPS production was restored to P. aeruginosa knockout mutants when the relevant B. pertussis genes were supplied in trans. Thus, the genes that encode the putative oxidase, transaminase, N-acetyltransferase, and epimerase enzymes in B. pertussis are functional homologs of those in P. aeruginosa. Two candidate dehydrogenase genes were located by searching the B. pertussis genome; these have 80% identity to P. aeruginosa wbpO (serotype O6) and 32% identity to wbpA (serotype O5). These genes, wbpO(1629) and wbpO(3150), were shown to complement a wbpA knockout of P. aeruginosa. Capillary electrophoresis was used to characterize the enzymatic activities of purified WbpO(1629) and WbpO(3150), and mass spectrometry analysis confirmed that the two enzymes are dehydrogenases capable of converting UDP-D-GlcNAc, UDP-D-GalNAc, to a lesser extent, and UDP-D-Glc, to a much lesser extent. Together, these results suggest that B. pertussis produces UDP-D-ManNAc3NAcA through the same pathway proposed for P. aeruginosa, despite differences in the genomic context of the genes involved.

Biosynthesis of UDP-N-acetyl-l-fucosamine, a Precursor to the Biosynthesis of Lipopolysaccharide in Pseudomonas aeruginosa Serotype O11

UDP-N-acetyl-L-fucosamine is a precursor to l-fucosamine in the lipopolysaccharide of Pseudomonas aeruginosa serotype O11 and the capsule of Staphylococcus aureus type 5. We have demonstrated previously the involvement of three enzymes, WbjB, WbjC, and WbjD, in the biosynthesis of UDP-2-acetamido-2,6-dideoxy-L-galactose or UDP-N-acetyl-L-fucosamine (UDP-l-FucNAc). An intermediate compound from the coupled-reaction of WbjB-WbjC with the initial substrate UDP-2-acetamido-2-deoxy-alpha-D-glucose or UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) was purified, and the structure was determined by NMR spectroscopy to be UDP-2-acetamido-2,6-dideoxy-L-talose (UDP-L-PneNAc). WbjD could then convert this intermediate into a new product with the same mass, consistent with a C-2 epimerization reaction. Those results led us to propose a pathway for the biosynthesis of UDP-L-FucNAc; however, the exact enzymatic activity of each of these proteins has not been defined. Here, we describe a fast protein liquid chromatography (FPLC)-based anion-exchange procedure, which allowed the separation and purification of the products of C-2 epimerization due to WbjD. Also, the application of a cryogenically cooled probe in NMR spectrometry offers the greatest sensitivity for determining the structures of minute quantities of materials, allowing the identification of the final product of the pathway. Our results showed that WbjB is bifunctional, catalyzing firstly C-4, C-6 dehydration and secondly C-5 epimerization in the reaction with the substrate UDP-D-GlcNAc, producing two intermediates. WbjC is also bifunctional, catalyzing C-3 epimerization of the second intermediate followed by reduction at C-4. The FPLC-based procedure provided good resolution of the final product of WbjD reaction from its epimer/substrate UDP-l-PneNAc, and the use of the cryogenically cooled probe in NMR revealed unequivocally that the final product is UDP-L-FucNAc.

Both myo-inositol to chiro-inositol epimerase activities and chiro-inositol to myo-inositol ratios are decreased in tissues of GK type 2 diabetic rats compared to Wistar controls

Previous data from our and other labs demonstrated a decreased chiro-inositol content in urine and tissues of human subjects and animals with type 2 diabetes. In urine this decrease in chiro-inositol was accompanied by an increase in myo-inositol content. Decreased urine levels of chiro-inositol in monkeys were next correlated with the severity of underlying insulin resistance determined by five separate assays. To investigate the decreased chiro-inositol and the accompanying increased myo-inositol excretions in urine in humans and monkeys, we postulated a defect in the epimerization of myo-inositol to chiro-inositol. [ 3H]Myo-inositol was then shown to be converted to [ 3H]chiro-inositol in rats in vivo and in fibroblasts in vitro in a process stimulated by insulin. We next demonstrated that the conversion of [ 3H]myo-inositol to [ 3H]chiro-inositol in vivo was markedly decreased in GK type 2 diabetic rats compared to Wistar controls in liver, muscle, and fat, insulin sensitive tissues. Decreases of 20–25% conversion to baseline levels of under 5% conversion were observed. In the present work, we initially compared the total contents of myo-inositol and chiro-inositol in GK type 2 diabetic rat kidney, liver, and muscle compared to Wistar controls. We demonstrated a consistent decreased total chiro-inositol to myo-inositol ratio in kidney, liver, and muscle compared to controls. We next established the presence of a myo-inositol to chiro-inositol epimerase activity in rat liver cytosol. Enzyme activity was shown to be time and enzyme concentration dependent with a broad pH optimum. It required NADH and NADPH for full activity, which is compatible with its action via an oxido-reductive mechanism. Lastly, we demonstrated that the epimerase enzyme bioactivity was significantly decreased in muscle, liver, and fat cytosolic extracts of GK type 2 diabetic rats versus Wistar controls. Decreased myo-inositol to chiro-inositol epimerase activity may therefore play a role in explaining the decreased chiro-inositol to myo-inositol urine and tissue ratios observed here and in previous animal and human studies. Further it may also possibly play a role in the underlying insulin resistance.

Characterisation of a sugar epimerase enzyme involved in the biosynthesis of a vancomycin-group antibiotic

An enzyme involved in the biosynthesis of the 4-epi-vancosamine substituents of a vancomycin group antibiotic has been expressed and its role as a TDP-4-keto-6-deoxyglucose-3,5-epimerase demonstrated.

Typhoid immunisation.

Since the desired improvement in the sanitation and nealth facilities is not likely in most areas of the third world in the near future, the necessity for typhoid vaccination cannot be easily disputed. The mechanisms of immune response in typhoid, especially with regard to the local gut factors are poorly understood. Parental vaccines have been in use for over 80 yr, but controlled field trials establishing their efficacy were conducted barely two decades back. Several studies in endemic areas under the auspices of the World Health Organisation established success in providing 51 to 94 percent protection with parenteral typhoid vaccine. The immunity, however, is not as complete as with some viral or toxoid vaccines, and can be overcome by a large infective dose of S. typhi. The acetone-inactivated vaccine is superior to other vaccines. Two sub-cutaneous doses of 0.5 ml each are as effective as intradermal doses of 0.1 ml each. The intradermal administration causes less local and systemic reactions. A mutant strain of Salmonella typhi Ty 21 a, lacking UDP galactose epimerase enzyme showed promise as a live oral vaccine in volunteer and field trials. More work, however, is needed to establish its efficacy and practical utility. This vaccine may possibly be the most effective vaccine ever prepared for typhoid fever. oral killed vaccine has been shown to have no beneficial effect in controlled trials. New modalities in developing vaccines such as using Vi capsular polysaccharide antigen and other mutants of S. typhi are being worked out.

Reversal of UDP-galactose 4-epimerase deficiency of human leukocytes in culture.

Stimulation with phytohemagglutinin of the leukocytes from six of the seven known individuals with UDP-galactose 4-epimerase (= UDP-glucose 4-epimerase; EC 5.1.3.2) deficiency consistently resulted in the appearance of epimerase activity in the cultured cells. A long-term lymphoblast culture derived from one proband also contained an active epimerase enzyme. A comparison of the properties of this enzyme with those of epimerase produced by control lymphoblast lines revealed comparable Km values for UDP-galactose and NAD and identical behavior on polyacrylamide electrophoresis. However, a difference in the NAD requirement for heat stability at 40 degree provided some evidence for a structural defect in this enzyme. Possible explanations for the appearance of UDP-galactose 4-epimerase activity in stimulated lymphocytes include an increased rate of synthesis of a mutant enzyme and a derepression of an epimerase locus during lymphocyte transformation.

Partial purification and kinetic properties of ribose-5-phosphate ketol-isomerase and ribulose-5-phosphate 3-epimerase from various sources

d-Ribose-5-phosphate ketol-isomerase (EC 5.3.1.6) was partially purified from calf spleen, calf liver and ox muscle. d-Ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) was prepared from calf liver. Mixtures of the isomerase and epimerase enzymes were separated by preparative electrophoresis in a ‘Pevikon’ block. Initial velocities of the isomerase and epimerase reactions were measured at 290 nm, at 340 nm, and by the cysteine carbazole method. The K m values for isomerase from spinach, spleen, muscle, and liver and for the epimerase from yeast and liver were determined. For spinach isomerase the K m values and the maximal velocities of both the forward and the reverse directions were measured.

Enzyme kinetics in mammalian cells. 3. Regulation of activities of galactokinase, galactose-1-phosphate uridyl transferase and uridine diphosphogalactose-4-epimerase in human erythrocytes.

AbstractThe sequential enzyme assay as previously described has been used to study various effects on the three enzymes in human red cells involved in the phosphorylation of galactose: galactokinase, galactose‐1‐phosphate uridyl transferase and uridine diphospho‐galactose‐4‐epimerase. Enzyme activities in undiluted lysates appear to reflect the respective activities in whole cells. Added extracellular Gal‐1‐P, G‐1‐P, UDPGal and UPDG do not affect enzyme activities in whole cells. The kinase and transferase enzymes do not appear to be associated with the membrane fraction of the red cells. Galactokinase activity is inhibited by G‐6‐P and Gal‐1‐P, but not by glucose, G‐1‐P, UDPG, UDPGal, UTP or NAD+. It is inhibited by ATP and ADP in high concentration. Galactose‐1‐phosphate uridyl transferase activity is inhibited by G‐1‐P, G‐6‐P, UDPG, UDPGal, ATP, and ADP. It is not affected by UTP, NAD+, or galactose. Uridine diphospho‐galactose‐4‐epimerase activity is inhibited by UDPG, ATP, ADP, UTP and NADH. It is stimulated by NAD+ and possibly by Gal‐1‐P. It is unaffected by G‐1‐P, G‐6‐P. The rates of the three reactions decrease with decreasing temperature. The activities of transferase and epimerase are inactivated at the same rate, the kinase activity is inactivated more slowly. Dilution experiments indicate the presence in lysates of a pool of UDPG (or, possibly UDPGal) which regulates the activities transferase and the epimerase enzymes. Results of dilution experiments suggest that the radioactive product of the transferase enzyme is different from commercially available UDPGal‐u‐14C. ATP, UTP and UDPG interact with some substance(s) in the red cell lysate to cause a time dependent inactivation of the epimerase. These interactions are the result of glucose metabolism.

Carbohydrate metabolism of lactic acid cultures. 3. Glycolytic enzymes of Streptococcus lactis and their sensitivity to antibiotics.

Streptococcus lactis UN possessed galactokinase, uridine diphosphate glucose epimerase (galacto-waldenase), hexokinase, aldolase, lactic dehydrogenase, alcohol dehydrogenase, and aldehyde dehydrogenase enzyme systems. The presence of galactokinase, galactowaldenase, and hexokinase indicated that monosaccharides, galactose, and glucose were phosphorylated and converted into glucose-6-phosphate, which then enters the glycolytic cycle. Galactokinase and UDPG epimerase enzymes were found to be adaptive in nature. However, hexokinase, aldolase, and dehydrogenases for lactate, alcohol, and aldehyde were found to be constitutive in nature, as the organism, regardless of the substrate used, always possessed these five enzymes. Both alcohol and aldehyde dehydrogenases were NAD-specific rather than NADP-specific.Penicillin, streptomycin, aureomycin, and terramycin inhibited the activities of these enzyme systems to varying degrees. Also, penicillin and streptomycin inhibited the production of galactokinase and UDPG epimerase by the organism, but had no effect upon the production of hexokinase.

LINGCOD MUSCLE PHOSPHORIBOISOMERASE AND RIBULOSE 5′-PHOSPHATE 3′-EPIMERASE

Comparatively pure phosphoriboisomerase and ribulose 5′-phosphate 3′-epimerase enzyme preparations were obtained from lingcod muscle by a simple procedure involving water extraction, saturation of the extract with ammonium sulphate, dialysis, brief heating to 55 °C, lyophilization of the solution, and final separation by ion exchange chromatography, using diethylamiuoethyl cellulose columns. Both enzymes have broad pH optima above pH 7.0, but are rapidly inactivated below this value. The following equilibria were established and compared with those obtained by other investigators: [Formula: see text], 1.35:1.0; [Formula: see text], 1: 1.5 and [Formula: see text], 1:0.58:0.66. The ketopentulose phosphate resulting from the action of phosphoriboisomerase on D-ribose 5-phosphate was isolated and identified as D-ribulose5-phosphate. Both D-ribulose and D-xylulose were demonstrated after subjecting a product of epimerase action to hydrolysis by acid phosphatase and ion exchange chromatography.

LINGCOD MUSCLE PHOSPHORIBOISOMERASE AND RIBULOSE 5′-PHOSPHATE 3′-EPIMERASE

Comparatively pure phosphoriboisomerase and ribulose 5′-phosphate 3′-epimerase enzyme preparations were obtained from lingcod muscle by a simple procedure involving water extraction, saturation of the extract with ammonium sulphate, dialysis, brief heating to 55 °C, lyophilization of the solution, and final separation by ion exchange chromatography, using diethylamiuoethyl cellulose columns. Both enzymes have broad pH optima above pH 7.0, but are rapidly inactivated below this value. The following equilibria were established and compared with those obtained by other investigators: [Formula: see text], 1.35:1.0; [Formula: see text], 1: 1.5 and [Formula: see text], 1:0.58:0.66. The ketopentulose phosphate resulting from the action of phosphoriboisomerase on D-ribose 5-phosphate was isolated and identified as D-ribulose5-phosphate. Both D-ribulose and D-xylulose were demonstrated after subjecting a product of epimerase action to hydrolysis by acid phosphatase and ion exchange chromatography.