المؤلفون: Michael T. Bowen, James R Krycer, Charlotte Fletcher, Lewis J. Martin, Jonathon C. Arnold, Marika Heblinski, Iain S. McGregor, Lyndsey L. Anderson, Elizabeth A. Cairns

المصدر: Journal of Natural Products. 84:1469-1477

مصطلحات موضوعية: In silico, Lactate dehydrogenase A, Pharmaceutical Science, 01 natural sciences, Cofactor, Analytical Chemistry, Cannabichromene, chemistry.chemical_compound, Lactate dehydrogenase, Drug Discovery, Binding site, education, Pharmacology, Virtual screening, education.field_of_study, biology, 010405 organic chemistry, Chemistry, Organic Chemistry, Ligand (biochemistry), 0104 chemical sciences, 010404 medicinal & biomolecular chemistry, Complementary and alternative medicine, Biochemistry, biology.protein, Molecular Medicine

الوصف: Cannabis sativa contains >120 phytocannabinoids, but our understanding of these compounds is limited. Determining the molecular modes of action of the phytocannabinoids may assist in their therapeutic development. Ligand-based virtual screening was used to suggest novel protein targets for phytocannabinoids. The similarity ensemble approach, a virtual screening tool, was applied to target identification for the phytocannabinoids as a class and predicted a possible interaction with the lactate dehydrogenase (LDH) family of enzymes. In order to evaluate this in silico prediction, a panel of 18 phytocannabinoids was screened against two LDH isozymes (LDHA and LDHB) in vitro. Cannabichromene (CBC) and Δ9-tetrahydrocannabinolic acid (Δ9-THCA) inhibited LDHA via a noncompetitive mode of inhibition with respect to pyruvate, with Ki values of 8.5 and 6.5 μM, respectively. In silico modeling was then used to predict the binding site for CBC and Δ9-THCA. Both were proposed to bind within the nicotinamide pocket, overlapping the binding site of the cofactor NADH, which is consistent with the noncompetitive modes of inhibition. Stemming from our in silico screen, CBC and Δ9-THCA were identified as inhibitors of LDHA, a novel molecular target that may contribute to their therapeutic effects.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::04f519a2fc8638f2e8daaa9ae7495df1Test
https://doi.org/10.1021/acs.jnatprod.0c01281Test

المؤلفون: Robin Dorau, Christian Solem, Jianming Liu, Peter Ruhdal Jensen, Ge Zhao, Jie Zhao

المصدر: Journal of Agricultural and Food Chemistry. 69:2826-2835

مصطلحات موضوعية: 0106 biological sciences, 01 natural sciences, Cofactor, chemistry.chemical_compound, Lactate dehydrogenase, polycyclic compounds, Food science, Nisin, Strain (chemistry), biology, Acetoin, 010401 analytical chemistry, Lactococcus lactis, food and beverages, General Chemistry, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, 0104 chemical sciences, Lactic acid, chemistry, biology.protein, bacteria, lipids (amino acids, peptides, and proteins), Fermentation, General Agricultural and Biological Sciences, 010606 plant biology & botany

الوصف: Nisin is commonly used as a biopreservative in foods. For industrial production, nisin-producing Lactococcus lactis strains are usually grown to high cell densities to achieve the highest possible nisin titer. However, accumulation of lactic acid eventually halts production, even in pH-controlled fermentations. Here, we describe a nisin-producing L. lactis strain Ge001, which was obtained after transferring the nisin gene cluster from L. lactis ATCC 11454, by conjugation, into the natural mutant L. lactis RD1M5, with low lactate dehydrogenase activity. The ability of Ge001 to produce nisin was tested using dairy waste as the fermentation substrate. To accommodate redox cofactor regeneration, respiration conditions were used, and to alleviate oxidative stress and to reduce adsorption of nisin onto the producing cells, we found it to be beneficial to add 1 mM Mn2+ and 100 mM Ca2+, respectively. A high titer of 12 084 IU/mL nisin could be reached, which is comparable to the highest titers reported using expensive, rich media. Summing up, we here present a 100% natural, robust, and sustainable approach for producing food-grade nisin and acetoin from readily available dairy waste.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::652d15cb4b7f5615c7d912747b094bc0Test
https://doi.org/10.1021/acs.jafc.0c07816Test

المؤلفون: Karin Müller-Decker, Jens Timmer, Christian Tönsing, Nese Erdem, Ulrike Korf, Stefan Wiemann, Devina Mitra, Stephan Bernhardt, Clemens Kreutz

المصدر: Journal of Proteome Research. 18:1352-1362

مصطلحات موضوعية: Proteomics, 0301 basic medicine, Proteome, Lactate dehydrogenase A, Breast Neoplasms, medicine.disease_cause, Biochemistry, Mice, 03 medical and health sciences, Breast cancer, Glutamate-Ammonia Ligase, medicine, Animals, Humans, education, Glucose Transporter Type 1, education.field_of_study, L-Lactate Dehydrogenase, 030102 biochemistry & molecular biology, biology, Effector, Glucose transporter, General Chemistry, Metabolism, Hypoxia (medical), Hypoxia-Inducible Factor 1, alpha Subunit, medicine.disease, Gene Expression Regulation, Neoplastic, Oxygen, 030104 developmental biology, MCF-7 Cells, Cancer research, biology.protein, Heterografts, Tumor Hypoxia, Female, GLUT1, medicine.symptom, Carcinogenesis

الوصف: Hypoxia as well as metabolism are central hallmarks of cancer, and hypoxia-inducible factors (HIFs) and metabolic effectors are crucial elements in oxygen-compromised tumor environments. Knowledge of changes in the expression of metabolic proteins in response to HIF function could provide mechanistic insights into adaptation to hypoxic stress, tumorigenesis, and disease progression. We analyzed time-resolved alterations in metabolism-associated protein levels in response to different oxygen potentials across breast cancer cell lines. Effects on the cellular metabolism of both HIF-dependent and -independent processes were analyzed by reverse-phase protein array profiling and a custom statistical model. We revealed a strong induction of glucose transporter 1 (GLUT1) and lactate dehydrogenase A (LDHA) as well as reduced glutamate-ammonia ligase (GLUL) protein levels across all cell lines tested as consistent changes upon hypoxia induction. Low GLUL protein levels were correlated with aggressive molecular subtypes in breast cancer patient data sets and also with hypoxic tumor regions in a xenograft mouse tumor model. Moreover, low GLUL expression was associated with poor survival in breast cancer patients and with high HIF-1α-expressing patient subgroups. Our data reveal time-resolved changes in the regulation of metabolic proteins under oxygen-deprived conditions and elucidate GLUL as a strong responder to HIFs and the hypoxic environment.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::9360fa9072116372abfd5f21c5ddb59aTest
https://doi.org/10.1021/acs.jproteome.8b00944Test

المؤلفون: Shunsuke Kobayashi, Akihiko Kondo, Kenji Takahashi, Hideo Kawaguchi, Tomokazu Shirai, Yota Tsuge, Kazuaki Ninomiya

المصدر: ACS Synthetic Biology. 9:814-826

مصطلحات موضوعية: 0106 biological sciences, 0303 health sciences, biology, Chemistry, Biomedical Engineering, General Medicine, Pentose phosphate pathway, 01 natural sciences, Biochemistry, Genetics and Molecular Biology (miscellaneous), Cofactor, Corynebacterium glutamicum, Metabolic engineering, 03 medical and health sciences, Metabolic pathway, chemistry.chemical_compound, Biochemistry, 010608 biotechnology, Lactate dehydrogenase, biology.protein, Glycolysis, Fermentation, 030304 developmental biology

الوصف: Controlling the carbon flux into a desired pathway is important for improving product yield in metabolic engineering. After entering a cell, glucose is channeled into glycolysis and the pentose phosphate pathway (PPP), which decreases the yield of target products whose synthesis relies on NADPH as a cofactor. Here, we demonstrate redirection of carbon flux into PPP under aerobic conditions in Corynebacterium glutamicum, achieved by replacing the promoter of glucose 6-phosphate isomerase gene (pgi) with an anaerobic-specific promoter of the lactate dehydrogenase gene (ldhA). The promoter replacement increased the split ratio of carbon flux into PPP from 39 to 83% under aerobic conditions. The titer, yield, and production rate of 1,5-diaminopentane, whose synthesis requires NADPH as a cofactor, were increased by 4.6-, 4.4-, and 2.6-fold, respectively. This is the largest improvement in the production of 1,5-diaminopentane or its precursor, lysine, reported to date. After aerobic cell growth, pgi expression was automatically induced under anaerobic conditions, altering the carbon flux from PPP to glycolysis, to produce succinate in a single metabolically engineered strain. Such an automatic redirection of metabolic pathway using an oxygen-responsive switch enables two-stage fermentation for efficient production of two different compounds by a single strain, potentially reducing the production costs and time for practical applications.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::37f74e8f6e37659c99809dede86cfcaeTest
https://doi.org/10.1021/acssynbio.9b00493Test

المؤلفون: Sergei Khrapunov, Robert Callender, Eric C. Chang

المصدر: Biochemistry. 56:3587-3595

مصطلحات موضوعية: 0301 basic medicine, Protein Denaturation, Protein Conformation, Swine, Biochemistry, Article, Substrate Specificity, Methylamines, 03 medical and health sciences, chemistry.chemical_compound, Lactate dehydrogenase, Enzyme Stability, Animals, Glycolysis, Psychrophile, chemistry.chemical_classification, L-Lactate Dehydrogenase, 030102 biochemistry & molecular biology, biology, Active site, NAD, Adaptation, Physiological, Perciformes, 030104 developmental biology, Enzyme, chemistry, Osmolyte, biology.protein, Thermodynamics, Lactate dehydrogenases, Mesophile

الوصف: The thermodynamics of substrate binding and enzymatic activity of a glycolytic enzyme, lactate dehydrogenase (LDH), from both porcine heart, phLDH (Sus scrofa; a mesophile), and mackerel icefish, cgLDH (Chamapsocephalus gunnari; a psychrophile), were investigated. Using a novel and quite sensitive fluorescence assay that can distinguish protein conformational changes close to and distal from the substrate binding pocket, a reversible global protein structural transition preceding the high-temperature transition (denaturation) was surprisingly found to coincide with a marked change in enzymatic activity for both LDHs. A similar reversible structural transition of the active site structure was observed for phLDH but not for cgLDH. An observed lower substrate binding affinity for cgLDH compared to that for phLDH was accompanied by a larger contribution of entropy to ΔG, which reflects a higher functional plasticity of the psychrophilic cgLDH compared to that of the mesophilic phLDH. The natural osmolyte, trimethylamine N-oxide (TMAO), increases stability and shifts all structural transitions to higher temperatures for both orthologs while simultaneously reducing catalytic activity. The presence of TMAO causes cgLDH to adopt catalytic parameters like those of phLDH in the absence of the osmolyte. Our results are most naturally understood within a model of enzyme dynamics whereby different conformations of the enzyme that have varied catalytic parameters (i.e., binding and catalytic proclivity) and whose population profiles are temperature-dependent and influenced by osmolytes interconvert among themselves. Our results also show that adaptation can be achieved by means other than gene mutations and complements the synchronic evolution of the cellular milieu.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::909acf5b616fb0ddd1c5316b83f53e5aTest
https://doi.org/10.1021/acs.biochem.7b00156Test

المؤلفون: Muhammad Aslam Shad, Young Ah Goo, Tanzila Rehman, Mazhar Hussain, David R. Goodlett, Mohd M. Khan, Benjamin L. Oyler

المصدر: Chemical Research in Toxicology. 29:1720-1728

مصطلحات موضوعية: 0301 basic medicine, Chromatography, biology, Arginine, Serum albumin, General Medicine, Toxicology, Blood proteins, 03 medical and health sciences, chemistry.chemical_compound, 030104 developmental biology, 0302 clinical medicine, Biochemistry, chemistry, Liquid chromatography–mass spectrometry, 030220 oncology & carcinogenesis, Lactate dehydrogenase, biology.protein, Alkaline phosphatase, Creatine kinase, Carbofuran

الوصف: This study was conducted to investigate the protein adducts with pesticides in a cohort of 172 factory workers that were exposed to a mixture of pesticides. The 35 samples showing considerable variation in biochemical parameters, i.e., butyrylcholinestrase (BChE), serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), gamma-glutamyl transferase (GGT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP/ALKP), lactate dehydrogenase (LDH), creatine phosphokinase (CPK) enzymes, and controls were analyzed by reversed-phase nanoscale liquid chromatography tandem mass spectrometry (nLC-MS/MS) on an Orbitrap mass spectrometer employing a shotgun proteomics approach. Only protein adducts with carbofuran were found on serum proteins of these workers. These adducts were of carbofuran labeled lysine (Lys-142, Lys-183, Lys-287, and Lys-467), arginine (Arg-210, Arg-242, and Arg-256) from serum albumin, and serine (Ser-07, Ser-54, and Ser-150) from immunoglobulin proteins. The arginine residues (Arg-210, Arg-242, Arg-246, and Arg-434) from albumin were also found to be glycated in serum of workers showing a high level of glucose who also had glycated arginine (Arg-1120) modified with carbofuran in their tankyrase-1-binding protein. The number of tandem mass spectra of modified peptides increased with increasing time of exposure. This is the first report to demonstrate the presence of carbofuran-labeled albumin, immunoglobulin, and glycated arginine, which shows that lysine and arginine of human albumin and serine of immunoglobulin are covalently modified in the serum of workers that were occupationally exposed to carbofuran, and the modification is detectable by tandem mass spectrometry. These peptides modified with carbofuran can potentially be used as a biomarker of carbofuran exposure.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::7461f906daeff675b06987abc5fcc8f5Test
https://doi.org/10.1021/acs.chemrestox.6b00222Test

المؤلفون: Xiaoliang Pan, Steven D. Schwartz

المصدر: The Journal of Physical Chemistry B. 119:5430-5436

مصطلحات موضوعية: Time Factors, Protein Conformation, Stereochemistry, Lactate dehydrogenase A, Context (language use), Molecular Dynamics Simulation, Nicotinamide adenine dinucleotide, Article, Cofactor, Enzyme catalysis, chemistry.chemical_compound, Lactate dehydrogenase, Pyruvic Acid, Materials Chemistry, Physical and Theoretical Chemistry, education, education.field_of_study, L-Lactate Dehydrogenase, biology, Energy landscape, NAD, Surfaces, Coatings and Films, Kinetics, chemistry, Biocatalysis, biology.protein, Thermodynamics, NAD+ kinase

الوصف: It has long been recognized that the structure of a protein creates a hierarchy of conformations interconverting on multiple time scales. The conformational heterogeneity of the Michaelis complex is of particular interest in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD(+)). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they show a strong variance in their propensity toward the on-enzyme chemical step. In this study, microsecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network of the Michaelis complex and the structures of the substates at atomistic scales. They also shed light on the complete picture of the catalytic mechanism of LDH.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::31be199ee1172943106ef71573bc4473Test
https://doi.org/10.1021/acs.jpcb.5b01840Test

المؤلفون: Linda S. Brunauer

المصدر: Journal of Chemical Education. 93:1108-1114

مصطلحات موضوعية: 0301 basic medicine, chemistry.chemical_classification, Bioanalysis, Chromatography, biology, Chemistry, Elution, education, 05 social sciences, Size-exclusion chromatography, 050301 education, General Chemistry, Enzyme assay, Education, 03 medical and health sciences, chemistry.chemical_compound, 030104 developmental biology, Enzyme, Affinity chromatography, Chemical engineering, Lactate dehydrogenase, Protein purification, biology.protein, 0503 education

الوصف: A multiweek protein purification suite, suitable for upper-division biochemistry or biotechnology undergraduate students, is described. Students work in small teams to isolate the enzyme lactate dehydrogenase (LDH) from a nontraditional tissue source, mammalian blood, using a sequence of three column chromatographic procedures: ion-exchange, size exclusion, and affinity chromatography. Protein and enzyme activity elution profiles are determined by graphical analysis of assay data collected using rapid microplate spectrophotometric assays. Students perform quantitative assays on LDH pools and use these data to build a purification table for use in evaluating the protocol. The protocol typically generates final overall fold-purifications from 1500 to 2500 and activity recoveries of 45–60%. Electrophoretic separations in both denaturing and native-gel format are analyzed both visually and by use of commercial digital analysis software to assess the isoenzyme pattern of isolated LDH and to further evaluate th...

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::1054ad53114adf45a65c2494f58ddb09Test
https://doi.org/10.1021/acs.jchemed.5b00713Test

المؤلفون: Hua Deng, Ruel Z. B. Desamero, Robert Callender, Beining Nie

المصدر: Biochemistry. 52:1886-1892

مصطلحات موضوعية: Models, Molecular, Protein Conformation, Kinetics, Mutant, Biochemistry, Article, Geobacillus stearothermophilus, chemistry.chemical_compound, Protein structure, Catalytic Domain, Lactate dehydrogenase, Point Mutation, chemistry.chemical_classification, Oxamic Acid, L-Lactate Dehydrogenase, biology, Temperature, Tryptophan, Wild type, Active site, NAD, Crystallography, Enzyme, chemistry, biology.protein, Biophysics

الوصف: Large scale dynamics within the Michaelis complex mimic of Bacillus stearothermophilus thermophilic lactate dehydrogenase, bsLDH·NADH·oxamate, were studied with site specific resolution by laser-induced temperature jump relaxation spectroscopy with a time resolution of 20 ns. NADH emission and Trp emission from the wild type and a series of single-tryptophan bsLDH mutants, with the tryptophan positions different distances from the active site, were used as reporters of evolving structure in response to the rapid change in temperature. Several distinct dynamical events were observed on the millisecond to microsecond time scale involving motion of atoms spread over the protein, some occurring concomitantly or nearly concomitantly with structural changes at the active site. This suggests that a large portion of the protein-substrate complex moves in a rather concerted fashion to bring about catalysis. The catalytically important surface loop undergoes two distinct movements, both needed for a competent enzyme. Our results also suggest that what is called "loop motion" is not just localized to the loop and active site residues. Rather, it involves the motion of atoms spread over the protein, even some quite distal from the active site. How these results bear on the catalytic mechanism of bsLDH is discussed.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::670aa23977066499fb4d48dfef9e21dcTest
https://doi.org/10.1021/bi3017125Test

المؤلفون: Rudolph E. Tanzi, Nianli Sang, Ramesh Raghupathi, Can Zhang, Haizhi Wang, Aleister J. Saunders

المصدر: Biochemistry

مصطلحات موضوعية: Leupeptins, Cathepsin L, Proteolysis, Protein degradation, Biochemistry, Article, Amyloid beta-Protein Precursor, Cell Line, Tumor, medicine, Amyloid precursor protein, Animals, Humans, Cells, Cultured, Cathepsin, L-Lactate Dehydrogenase, medicine.diagnostic_test, biology, Calpain, Chemistry, Ubiquitination, Peptide Fragments, Rats, Alpha secretase, biology.protein, Cattle, Amyloid Precursor Protein Secretases, Amyloid precursor protein secretase

الوصف: Alzheimer's disease (AD) is characterized by the deposition of amyloid β (Aβ), a peptide generated from proteolytic processing of its precursor, amyloid precursor protein (APP). Canonical APP proteolysis occurs via α-, β-, and γ-secretases. APP is also actively degraded by protein degradation systems. By pharmacologically inhibiting protein degradation with ALLN, we observed an accumulation of several novel APP C-terminal fragments (CTFs). The two major novel CTFs migrated around 15 and 25 kDa and can be observed across multiple cell types. The process was independent of cytotoxicity or protein synthesis. We further determine that the accumulation of the novel CTFs is not mediated by proteasome or calpain inhibition, but by cathepsin L inhibition. Moreover, these novel CTFs are not generated by an increased amount of BACE. Here, we name the CTF of 25 kDa as η-CTF (eta-CTF). Our data suggest that under physiological conditions, a subset of APP undergoes alternative processing and the intermediate products, the 15 kDa CTFs, and the η-CTFs aret rapidly degraded and/or processed via the protein degradation machinery, specifically, cathepsin L.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::b874b95c92707fe918d58934d93328d8Test
https://doi.org/10.1021/acs.biochem.5b00329Test