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The Journal of Experimental Biology Jun 2015Sensitivity to temperature helps determine the success of organisms in all habitats, and is caused by the susceptibility of biochemical processes, including enzyme... (Review)
Review
Sensitivity to temperature helps determine the success of organisms in all habitats, and is caused by the susceptibility of biochemical processes, including enzyme function, to temperature change. A series of studies using two structurally and catalytically related enzymes, A4-lactate dehydrogenase (A4-LDH) and cytosolic malate dehydrogenase (cMDH) have been especially valuable in determining the functional attributes of enzymes most sensitive to temperature, and identifying amino acid substitutions that lead to changes in those attributes. The results of these efforts indicate that ligand binding affinity and catalytic rate are key targets during temperature adaptation: ligand affinity decreases during cold adaptation to allow more rapid catalysis. Structural changes causing these functional shifts often comprise only a single amino acid substitution in an enzyme subunit containing approximately 330 residues; they occur on the surface of the protein in or near regions of the enzyme that move during catalysis, but not in the active site; and they decrease stability in cold-adapted orthologs by altering intra-molecular hydrogen bonding patterns or interactions with the solvent. Despite these structure-function insights, we currently are unable to predict a priori how a particular substitution alters enzyme function in relation to temperature. A predictive ability of this nature might allow a proteome-wide survey of adaptation to temperature and reveal what fraction of the proteome may need to adapt to temperature changes of the order predicted by global warming models. Approaches employing algorithms that calculate changes in protein stability in response to a mutation have the potential to help predict temperature adaptation in enzymes; however, using examples of temperature-adaptive mutations in A4-LDH and cMDH, we find that the algorithms we tested currently lack the sensitivity to detect the small changes in flexibility that are central to enzyme adaptation to temperature.
Topics: Adaptation, Physiological; Amino Acid Sequence; Amino Acid Substitution; Animals; L-Lactate Dehydrogenase; Malate Dehydrogenase; Molecular Sequence Data; Protein Conformation; Temperature
PubMed: 26085658
DOI: 10.1242/jeb.114298 -
Applied and Environmental Microbiology Jan 2021Growth of PCA on lactate was enhanced by laboratory adaptive evolution. The enhanced growth was considered to be attributed to increased expression of the genes,...
Growth of PCA on lactate was enhanced by laboratory adaptive evolution. The enhanced growth was considered to be attributed to increased expression of the genes, encoding a succinyl-coenzyme A (CoA) synthetase. To further investigate the function of the succinyl-CoA synthetase, the genes were deleted from The mutant showed defective growth on lactate but not on acetate. Introduction of the genes into the mutant restored the full potential to grow on lactate. These results verify the importance of the succinyl-CoA synthetase in growth on lactate. Genome analysis of species identified candidate genes, GSU1623, GSU1624, and GSU1620, for lactate dehydrogenase. Deletion mutants of the identified genes for d-lactate dehydrogenase (ΔGSU1623 ΔGSU1624 mutant) or l-lactate dehydrogenase (ΔGSU1620 mutant) could not grow on d-lactate or l-lactate but could grow on acetate and l- or d-lactate, respectively. Introduction of the respective genes into the mutants allowed growth on the corresponding lactate stereoisomer. These results suggest that the identified genes were essential for d- or l-lactate utilization. The reporter assay demonstrated that the putative promoter regions were more active during growth on lactate than during growth on acetate, indicating that the genes for the lactate dehydrogenases were expressed more during growth on lactate than during growth on acetate. The gene deletion phenotypes and the expression profiles indicate that there are metabolic switches between lactate and acetate. This study advances the understanding of anaerobic lactate utilization in Lactate is a microbial fermentation product as well as a source of carbon and electrons for microorganisms in the environment. Furthermore, lactate is a common amendment for stimulation of microbial growth in environmental biotechnology applications. However, anaerobic metabolism of lactate has been poorly studied for environmentally relevant microorganisms. species are found in various environments and environmental biotechnology applications. By employing genomic and genetic approaches, succinyl-CoA synthetase and lactate dehydrogenase were identified as key enzymes in anaerobic metabolism of lactate in , a representative species. Differential gene expression during growth on lactate and acetate was observed, demonstrating that could metabolically switch to adapt to available substrates in the environment. The findings provide new insights into basic physiology in lactate metabolism as well as cellular responses to growth conditions in the environment and can be informative for the application of lactate in environmental biotechnology.
Topics: Anaerobiosis; Bacterial Proteins; Gene Expression Regulation, Bacterial; Geobacter; L-Lactate Dehydrogenase; Lactic Acid; Succinate-CoA Ligases
PubMed: 33158892
DOI: 10.1128/AEM.01968-20 -
Protein Expression and Purification Sep 2017Shrimp lactate dehydrogenase (LDH) is induced in response to environmental hypoxia. Two protein subunits deduced from different transcripts of the LDH gene from the...
Shrimp lactate dehydrogenase (LDH) is induced in response to environmental hypoxia. Two protein subunits deduced from different transcripts of the LDH gene from the shrimp Litopenaeus vannamei (LDHvan-1 and LDHvan-2) were identified. These subunits are expressed by alternative splicing. Since both subunits are expressed in most tissues, the purification of the enzyme from the shrimp will likely produce hetero LDH containing both subunits. Therefore, the aim of this study was to overexpress, purify and characterize only one subunit as a recombinant protein, the LDHvan-2. For this, the cDNA from muscle was cloned and overexpressed in E. coli as a fusion protein containing an intein and a chitin binding protein domain (CBD). The recombinant protein was purified by chitin affinity chromatography column that retained the CBD and released solely the full and active LDH. The active protein appears to be a tetramer with molecular mass of approximately 140 kDa and can use pyruvate or lactate as substrates, but has higher specific activity with pyruvate. The enzyme is stable between pH 7.0 to 8.5, and between 20 and 50 °C with an optimal temperature of 50 °C. Two pK of 9.3 and 6.6, and activation energy of 44.8 kJ/mol°K were found. The kinetic constants K for NADH was 23.4 ± 1.8 μM, and for pyruvate was 203 ± 25 μM, while V was 7.45 μmol/min/mg protein. The shrimp LDH that is mainly expressed in shrimp muscle preferentially converts pyruvate to lactate and is an important enzyme for the response to hypoxia.
Topics: Animals; Arthropod Proteins; Escherichia coli; Gene Expression; L-Lactate Dehydrogenase; Penaeidae; Recombinant Proteins
PubMed: 28625911
DOI: 10.1016/j.pep.2017.06.010 -
Methods in Molecular Biology (Clifton,... 2020Apicomplexans are obligate parasites that replicate inside host cells, within a subcellular compartment called the parasitophorous vacuole. Egress is the process by...
Apicomplexans are obligate parasites that replicate inside host cells, within a subcellular compartment called the parasitophorous vacuole. Egress is the process by which apicomplexan parasites like Toxoplasma gondii exit from host cells, rupturing the parasitophorous vacuole and host-cell plasma membranes in the process. T. gondii retains the ability to egress throughout most of its intracellular replicative cycle, and this process has been associated with parasite signaling pathways that include the modulation of intracellular calcium, cyclic nucleotides, phosphatidic acid, and pH, which can be manipulated genetically or pharmacologically. Here we describe two methods of assessing stimulated parasite egress from host cells by measuring the permeabilization of host-cell membranes that occurs during this process. The first method measures the release of lactate dehydrogenase (LDH) from host cells, which is quantified in a colorimetric assay that detects LDH by the enzymatic generation of red formazan. The second method measures entry of the cell-impermeant 4',6-diamidino-2-phenylindole (DAPI) DNA dye, which stains host-cell nuclei (HCN) as parasites egress. Both described methods complement, with higher throughput, video-microscopy approaches that are well suited to examine the dissociation of parasite vacuoles that follows host-cell permeabilization.
Topics: Cell Nucleus; Colorimetry; Kinetics; L-Lactate Dehydrogenase; Toxoplasma
PubMed: 31758453
DOI: 10.1007/978-1-4939-9857-9_10 -
American Journal of Clinical Pathology Feb 2015
Topics: Humans; L-Lactate Dehydrogenase
PubMed: 25596240
DOI: 10.1309/AJCTP0FC8QFYDFA -
Orvosi Hetilap Dec 2017Glycolysis is increased in most of the malignant cells, providing the largest proportion of energy needed for cell proliferation. Lactate dehydrogenase (LDH) catalyses... (Review)
Review
Glycolysis is increased in most of the malignant cells, providing the largest proportion of energy needed for cell proliferation. Lactate dehydrogenase (LDH) catalyses the reversible process of pyruvate to lactate in anaerobic condition. LDHA isoenzyme expressed mainly by malignant cells, significantly increases lactate formation. Lactate induces the proliferation of oxygenated malignant cells, angiogenesis, and inhibits the innate and adaptive immune responses. Baseline serum LDH elevation correlates with shorter survival. The authors review the relevant studies exploring the correlation between LDH elevation and the prognosis of malignant diseases. Orv Hetil. 2017; 158(50): 1977-1988.
Topics: Biomarkers, Tumor; Female; Humans; L-Lactate Dehydrogenase; Medical Oncology; Prognosis; Urogenital Neoplasms
PubMed: 29226713
DOI: 10.1556/650.2017.30890 -
Biomedicine & Pharmacotherapy =... Mar 2022Cancer is one of the main causes of human mortality and brain tumors, including invasive pituitary adenomas, medulloblastomas and glioblastomas are common brain... (Review)
Review
Cancer is one of the main causes of human mortality and brain tumors, including invasive pituitary adenomas, medulloblastomas and glioblastomas are common brain malignancies with poor prognosis. Therefore, the development of innovative management strategies for refractory cancers and brain tumors is important. In states of mitochondrial dysfunction - commonly encountered in malignant cells - cells mostly shift to anaerobic glycolysis by increasing the expression of LDHA (Lactate Dehydrogenase-A) gene. Oxamate, an isosteric form of pyruvate, blocks LDHA activity by competing with pyruvate. By blocking LDHA, it inhibits protumorigenic cascades and also induces ROS (reactive oxygen species)-induced mitochondrial apoptosis of cancer cells. In preclinical studies, oxamate blocked the growth of invasive pituitary adenomas, medulloblastomas and glioblastomas. Oxamate also increases temozolomide and radiotherapy sensitivity of glioblastomas. Oxamate is highly polar, which may preclude its clinical utilization due to low penetrance through cell membranes. However, this obstacle could be overcome with nanoliposomes. Moreover, different oxamate analogs were developed which inhibit LDHC4, an enzyme also involved in cancer progression and germ cell physiology. Lastly, phenformin, an antidiabetic agent, exerts anticancer effects via complex I inhibition in the mitochondria and leading the overproduction of ROS. Oxamate combination with phenformin reduces the lactic acidosis-causing side effect of phenformin while inducing synergistic anticancer efficacy. In sum, oxamate as a single agent and more efficiently with phenformin has high potential to slow the progression of aggressive cancers with special emphasis to brain tumors.
Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Glycolysis; Humans; L-Lactate Dehydrogenase; Mitochondria; Neoplasms; Oxamic Acid; Phenformin; Radiation Tolerance; Reactive Oxygen Species; Temozolomide
PubMed: 35124385
DOI: 10.1016/j.biopha.2022.112686 -
Biochemical and Biophysical Research... Oct 2016Lactate dehydrogenase (LDH) is a glycolytic enzyme that catalyzes the final step of glycolysis and produces NAD. In somatic cells, LDH forms homotetramers and...
Lactate dehydrogenase (LDH) is a glycolytic enzyme that catalyzes the final step of glycolysis and produces NAD. In somatic cells, LDH forms homotetramers and heterotetramers that are encoded by two different genes: LDHA (skeletal muscle type, M) and LDHB (heart type, H). Analysis of LDH isozymes is important for understanding the physiological role of homotetramers and heterotetramers and for optimizing inhibition of their enzymatic activity as it may result in distinct effects. Previously, we reported that hydroxychloroquine (HCQ) inhibited LDH activity, but we did not examine isozyme specificity. In the present study, we isolated heterotetrameric LDH (HM) from swine brain, determined its kinetic and thermodynamic properties, and examined the effect of HCQ on its activity compared to homotetrameric LDH isozymes. We show that: (1) the K values for HM-mediated catalysis of pyruvate or lactate were intermediate compared to those for the homotetrameric isozymes, M and H whereas the V values were similar; (2) the K and V values for HM-mediated catalysis of NADH were not significantly different among LDH isozymes; (3) the values for activation energy and van't Hoff enthalpy changes for pyruvate reduction of HM were intermediate compared to those for the homotetrameric isozymes; (4) the temperature for half residual activity of HM was closer to that for M than for H. We also show that HCQ had different affinities for various LDH isozymes.
Topics: Animals; Brain; Enzyme Inhibitors; Hydroxychloroquine; Isoenzymes; Kinetics; L-Lactate Dehydrogenase; Protein Structure, Quaternary; Protein Subunits; Swine; Thermodynamics
PubMed: 27671200
DOI: 10.1016/j.bbrc.2016.09.118 -
Biomedicine & Pharmacotherapy =... Apr 2023Even though the pathophysiology of colorectal cancer (CRC) is complicated and poorly understood, interactions between risk factors appear to be key in the development... (Review)
Review
Even though the pathophysiology of colorectal cancer (CRC) is complicated and poorly understood, interactions between risk factors appear to be key in the development and progression of the malignancy. The popularity of using lactic acid bacteria (LAB) prebiotics and probiotics to modulate the tumor microenvironment (TME) has grown widely over the past decade. The objective of this study was therefore to determine the detrimental effects of LAB-derived lactic acid in the colonic mucosa in colorectal cancer management. Six library databases and a web search engine were used to execute a structured systematic search of the existing literature, considering all publications published up until August 2022. A total of 7817 papers were screened, all of which were published between 1995 and August 2022. However, only 118 articles met the inclusion criterion. Lactic acid has been directly linked to the massive proliferation of cancerous cells since the glycolytic pathway provides cancerous cells with not only ATP, but also biosynthetic intermediates for rapid growth and proliferation. Our research suggests that targeting LAB metabolic pathways is capable of suppressing tumor growth and that the LDH gene is critical for tumorigenesis. Silencing of Lactate dehydrogenase, A (LDHA), B (LDHB), (LDHL), and hicD genes should be explored to inhibit fermentative glycolysis yielding lactic acid as the by-product. More studies are necessary for a solid understanding of this topic so that LAB and their corresponding lactic acid by-products do not have more adverse effects than their widely touted positive outcomes in CRC management.
Topics: Humans; L-Lactate Dehydrogenase; Glycolysis; Lactic Acid; Colorectal Neoplasms; Probiotics; Tumor Microenvironment
PubMed: 36758316
DOI: 10.1016/j.biopha.2023.114371 -
Current Genetics Apr 2019Lactate dehydrogenase (LDH) widely exists in organisms, which catalyzes the interconversion of pyruvate into lactate with concomitant interconversion of NADH and NAD. In...
Lactate dehydrogenase (LDH) widely exists in organisms, which catalyzes the interconversion of pyruvate into lactate with concomitant interconversion of NADH and NAD. In this study, two L-type lactate dehydrogenase genes FgLDHL1 and FgLDHL2 were characterized in an ascomycete fungus Fusarium graminearum, a causal agent of wheat head blight. Both the single-gene deletion mutants of FgLDHL1 or FgLDHL2 exhibited phenotypic defects in vegetative growth, sporulation, spore germination, L-lactate biosynthesis and activity. Additionally, the two L-lactate dehydrogenases were involved in the utilization of carbon sources and maintenance of redox homeostasis during spore germination. Pathogenicity assays showed that ΔFgLDHL1 exhibits reduced virulence on wheat spikelets and on corn stigmas, suggesting that it was indirectly correlated with a reduced level of deoxynivalenol accumulation. These results indicate that FgLDHL1 and FgLDHL2 play multiple roles in the developmental processes and pathogenesis in F. graminearum, and help understand the functional diversity of D-/L-lactate dehydrogenase in phytopathogenic fungi.
Topics: Amino Acid Sequence; Environment; Fusarium; Genes, Fungal; Genetic Complementation Test; Hyphae; L-Lactate Dehydrogenase; Phenotype; Phylogeny; Plant Diseases; Sensitivity and Specificity; Sequence Analysis, DNA; Sequence Deletion; Spores, Fungal; Stress, Physiological
PubMed: 30474697
DOI: 10.1007/s00294-018-0909-6