Cloning and characterization of the Bambusa oldhamii BoMDH-encoded malate dehydrogenase.

Malate dehydrogenase (MDH), which is ubiquitously occurred in nature, catalyzes the interconversion of malate and oxaloacetate. Higher plants contain multiple forms of MDH that differ in coenzyme specificity, subcellular localization and physiological function. A putative Bambusa oldhamii BoMDH cDNA was screened with the specific probe from the bamboo cDNA library. Sequence alignment shows that there's a high homology between the deduced amino acid sequence of BoMDH and MDH protein in Oryza sativa glyoxysome (92%). A 57 kDa fusion protein was expressed by IPTG induction in Escherichia coli BL21 (DE3), and an obvious MDH activity was detected in the recombinant protein. The molecular mass of recombinant BoMDH was estimated to be 120 kDa, and the subunit form was 57 kDa by denatured SDS-PAGE, indicating that BoMDH presents as a homodimer. The optimum temperature and pH for BoMDH activity were 40 °C and 9.5, respectively. The Km values of BoMDH for malate and NAD+ were 5.2 mM and 0.52 mM. The kcat/Km values of BoMDH for malate and NAD+ were 163 min-1 mM-1 and 3060 min-1 mM-1.

A novel malic enzyme gene, Mime2, from Mortierella isabellina M6-22 contributes to lipid accumulation.

OBJECTIVE: This study was aimed at cloning and characterizing a novel malic enzyme (ME) gene of Mortierella isabellina M6-22 and identifying its relation with lipid accumulation. METHODS: Mime2 was cloned from strain M6-22. Plasmid pET32aMIME2 was constructed to express ME of MIME2 in Escherichia coli BL21. After purification, the optimal pH and temperature of MIME2, as well as Km and Vmax for NADP+ were determined. The effects of EDTA or metal ions (Mn2+, Mg2+, Co2+, Cu2+, Ca2+, or Zn2+) on the enzymatic activity of MIME2 were evaluated. Besides, plasmid pRHMIME2 was created to express MIME2 in Rhodosporidium kratochvilovae YM25235, and its cell lipid content was measured by the acid-heating method. The optimal pH and temperature of MIME2 are 5.8 and 30 °C, respectively. RESULTS: The act ivity of MIME2 was significantly increased by Mg2+, Ca2+, or Mn2+ at 0.5 mM but inhibited by Cu2+ or Zn2+ (p < 0.05). The optimal enzymatic activity of MIME2 is 177.46 U/mg, and the Km and Vmax for NADP+ are 0.703 mM and 156.25 µg/min, respectively. Besides, Mime2 transformation significantly increased the cell lipid content in strain YM25235 (3.15 ± 0.24 vs. 2.17 ± 0.31 g/L, p < 0.01). CONCLUSIONS: The novel ME gene Mime2 isolated from strain M6-22 contributes to lipid accumulation in strain YM25235.

Differential Protein Expressions in Virus-Infected and Uninfected Trichomonas vaginalis.

Protozoan viruses may influence the function and pathogenicity of the protozoa. Trichomonas vaginalis is a parasitic protozoan that could contain a double stranded RNA (dsRNA) virus, T. vaginalis virus (TVV). However, there are few reports on the properties of the virus. To further determine variations in protein expression of T. vaginalis, we detected 2 strains of T. vaginalis; the virus-infected (V+) and uninfected (V-) isolates to examine differentially expressed proteins upon TVV infection. Using a stable isotope N-terminal labeling strategy (iTRAQ) on soluble fractions to analyze proteomes, we identified 293 proteins, of which 50 were altered in V+ compared with V- isolates. The results showed that the expression of 29 proteins was increased, and 21 proteins decreased in V+ isolates. These differentially expressed proteins can be classified into 4 categories: ribosomal proteins, metabolic enzymes, heat shock proteins, and putative uncharacterized proteins. Quantitative PCR was used to detect 4 metabolic processes proteins: glycogen phosphorylase, malate dehydrogenase, triosephosphate isomerase, and glucose-6-phosphate isomerase, which were differentially expressed in V+ and V- isolates. Our findings suggest that mRNA levels of these genes were consistent with protein expression levels. This study was the first which analyzed protein expression variations upon TVV infection. These observations will provide a basis for future studies concerning the possible roles of these proteins in host-parasite interactions.

Purification and characterization of the plastid-localized NAD-dependent malate dehydrogenase from Arabidopsis thaliana.

Malate dehydrogenase (MDH) ubiquitously exists in living organisms and has many isoforms in a single species. MDHs from some classes have been characterized for their catalytic properties, which show significant variations despite that they share high sequence identity for the active sites. One class MDH, the plastid-localized NAD-dependent MDH (plNAD-MDH) is known to be important for plant survival in a dark environment, but its biochemical and enzymatic properties have not been well characterized. This study attempts to fill the gap. plNAD-MDH was expressed in an Escherichia coli system and purified using nickel-affinity chromatography followed by size exclusion chromatography. The N-terminal fusion his-tag was removed by protease cleavage. The gel filtration assay and glutaraldehyde cross-linking results showed that the active enzyme was a homodimer in solution. Further assay indicated that plNAD-MDH is most active at a neutral pH value. The Km values for oxaloacetate and NADH are found in the submillimolar order, a median range for most MDHs. The maximum reaction rate values, however, are dramatically different from other plant MDHs, indicating that plNAD-MDH has different substrate specificity. Moreover, we obtained crystals for this enzyme, which laid the groundwork for further analysis of the enzymatic mechanism from structural stand point.

Trypanosoma evansi contains two auxiliary enzymes of glycolytic metabolism: Phosphoenolpyruvate carboxykinase and pyruvate phosphate dikinase.

Trypanosoma evansi is a monomorphic protist that can infect horses and other animal species of economic importance for man. Like the bloodstream form of the closely related species Trypanosoma brucei, T. evansi depends exclusively on glycolysis for its free-energy generation. In T. evansi as in other kinetoplastid organisms, the enzymes of the major part of the glycolytic pathway are present within organelles called glycosomes, which are authentic but specialized peroxisomes. Since T. evansi does not undergo stage-dependent differentiations, it occurs only as bloodstream forms, it has been assumed that the metabolic pattern of this parasite is identical to that of the bloodstream form of T. brucei. However, we report here the presence of two additional enzymes, phosphoenolpyruvate carboxykinase and PPi-dependent pyruvate phosphate dikinase in T. evansi glycosomes. Their colocalization with glycolytic enzymes within the glycosomes of this parasite has not been reported before. Both enzymes can make use of PEP for contributing to the production of ATP within the organelles. The activity of these enzymes in T. evansi glycosomes drastically changes the model assumed for the oxidation of glucose by this parasite.

[Physicochemical and catalytic properties of NAD+- dependent malate dehydrogenase isoforms from maize mesophyll0.

Malate dehyrogenase isoforms (46- and 70-fold purifications) with specific activities of the 640 and 990 U/mg protein were obtained in an electrophoretically homogeneous state from maize mesophyll. The physicochemical and catalytic properties of these isoforms were studied. The molecular weight and the Michaelis constants were determined; the effect of hydrogen ions on the forward and reverse MDH reaction was studied. The results of SDS-PAGE demonstrated that malate dehydrogenase isoforms have an oligomeric structure comprised of identical subunits. The first isoform with a molecular weight of 126.58 kDa is tetramer, and the second isoform with a molecular weight of 63.3 is dimer.

[Isoformes of Malate Dehydrogenase from Rhodovulum Steppense A-20s Grown Chemotrophically under Aerobic Condtions].

Three malate dehydrogenase isoforms (65-, 60-, and 71-fold purifications) with specific activities of 4.23, 3.88, and 4.56 U/mg protein were obtained in an electrophoretically homogenous state from Rhodovulum steppense bacteria strain A-20s chemotropically grown under aerobic conditions. The physicochemical and kinetic properties of malate dehydrogenase isoforms were determined. The molecular weight and the Michaelis constants were determined; the effect of hydrogen ions on the forward and reverse MDH reaction was studied. The results of the study demonstrated that the enzyme consists of subunits; the molecular weight of subunits was determined by SDS-PAGE.

Reassessment of the transhydrogenase/malate shunt pathway in Clostridium thermocellum ATCC 27405 through kinetic characterization of malic enzyme and malate dehydrogenase.

Clostridium thermocellum produces ethanol as one of its major end products from direct fermentation of cellulosic biomass. Therefore, it is viewed as an attractive model for the production of biofuels via consolidated bioprocessing. However, a better understanding of the metabolic pathways, along with their putative regulation, could lead to improved strategies for increasing the production of ethanol. In the absence of an annotated pyruvate kinase in the genome, alternate means of generating pyruvate have been sought. Previous proteomic and transcriptomic work detected high levels of a malate dehydrogenase and malic enzyme, which may be used as part of a malate shunt for the generation of pyruvate from phosphoenolpyruvate. The purification and characterization of the malate dehydrogenase and malic enzyme are described in order to elucidate their putative roles in malate shunt and their potential role in C. thermocellum metabolism. The malate dehydrogenase catalyzed the reduction of oxaloacetate to malate utilizing NADH or NADPH with a kcat of 45.8 s(-1) or 14.9 s(-1), respectively, resulting in a 12-fold increase in catalytic efficiency when using NADH over NADPH. The malic enzyme displayed reversible malate decarboxylation activity with a kcat of 520.8 s(-1). The malic enzyme used NADP(+) as a cofactor along with NH4 (+) and Mn(2+) as activators. Pyrophosphate was found to be a potent inhibitor of malic enzyme activity, with a Ki of 0.036 mM. We propose a putative regulatory mechanism of the malate shunt by pyrophosphate and NH4 (+) based on the characterization of the malate dehydrogenase and malic enzyme.

Superior aluminium (Al) tolerance of Stylosanthes is achieved mainly by malate synthesis through an Al-enhanced malic enzyme, SgME1.

Stylosanthes (stylo) is a dominant leguminous forage in the tropics. Previous studies suggest that stylo has great potential for aluminium (Al) tolerance, but little is known about the underlying mechanism. A novel malic enzyme, SgME1, was identified from the Al-tolerant genotype TPRC2001-1 after 72 h Al exposure by two-dimensional electrophoresis, and the encoding gene was cloned and characterized via heterologous expression in yeast, Arabidopsis thaliana and bean (Phaseolus vulgaris) hairy roots. Internal Al detoxification might be mainly responsible for the 72 h Al tolerance of TPRC2001-1, as indicated by 5.8-fold higher root malate concentrations and approximately two-fold higher Al concentrations in roots and root symplasts of TPRC2001-1 than those of the Al-sensitive genotype Fine-stem. An accompanying increase in malate secretion might also reduce a fraction of Al uptake in TPRC2001-1. Gene and protein expression of SgME1 was only enhanced in TPRC2001-1 after 72 h Al exposure. Overexpressing SgME1 enhanced malate synthesis and rescued yeast, A. thaliana and bean hairy roots from Al toxicity via increasing intracellular malate concentrations and/or accompanied malate exudation. These results provide strong evidence that superior Al tolerance of stylo is mainly conferred by Al-enhanced malate synthesis, functionally controlled by SgME1.

Molecular analysis of cytosolic and mitochondrial malate dehydrogenases isolated from domestic cats (Felis catus).

Malate dehydrogenase (MDH) plays crucial roles in energy and cellular metabolism. In this study, we describe the identification and characterization of cytosolic MDH (MDH1) and mitochondrial MDH (MDH2) in liver of domestic cat (Felis catus). To clone the feline full-length MDH genes, we performed rapid amplification of cDNA ends. The MDH1 gene encoded a protein of 334 amino acids and the MDH2 gene encoded a protein of 338 amino acids, containing a 24-amino acid mitochondrial target sequence. The feline MDH1 and MDH2 proteins shared, respectively, 98.8-93.7 and 96.7-94.4% homology with dog, giant panda, horse, cow, pig, human, mouse, and rat. The feline MDHs had a highly conserved active motif, which contained important residues for catalysis and coenzyme binding. The putatively acetylated lysine residues that regulate MDH activity were also conserved at K118, K121, and K298 in MDH1, and K185, K301, K307, and K314 in MDH2. Both MDH1 and MDH2 mRNAs were ubiquitously expressed, but these expression levels varied in a tissue-specific manner. Both MDH genes were expressed at considerably high levels in heart and skeletal muscle, but at low levels in lung and spleen.

[Physicochemical, catalytic, and regulatory properties of malate dehydrogenase from Rhodovulum steppense bacteria, strain A-20s].

The physicochemical, regulatory, and kinetic properties of malate dehydrogenase (EC 1.1.1.37) from haloalkaliphilic purple nonsulfur Rhodovulum steppense bacteria, strain A-20s, were studied. The malate dehydrogenase (MDH) preparation with a specific activity of 0.775 ± 0.113 U/mg protein was obtained in an electrophoretically homogeneous state using multistep purification. Using homogenous preparations, the molecular weight and the Michaelis constant of the enzyme were determined; the effects of metal ions, the temperature effect, and the thermal stability of the MDH were studied. The dimer structure of the enzyme was demonstrated by DS-Na-electrophoresis.

Cloning, overexpression, purification and crystallization of malate dehydrogenase from Thermus thermophilus.

Malate dehydrogenase (MDH) has been used as a conjugate for enzyme immunoassay of a wide variety of compounds, such as drugs of abuse, drugs used in repetitive therapeutic application and hormones. In consideration of the various biotechnological applications of MDH, investigations of MDH from Thermus thermophilus were carried out to further understand the properties of this enzyme. The DNA fragment containing the open reading frame of mdh was amplified from the genomic DNA of T. thermophilus and cloned into the expression vector pET21b(+). The protein was expressed in a soluble form in Escherichia coli strain BL21(DE3). Homogeneous protein was obtained using a three-step procedure consisting of thermal treatment, Ni(2+)-chelating chromatography and size-exclusion chromatography. The purified MDH was crystallized and the crystals diffracted to a resolution of 1.80 Šon the BL13C1 beamline of the National Synchrotron Radiation Research Center (NSRRC), Taiwan. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 71.3, b = 86.1, c = 118.2 Å. The unit-cell volume of the crystal is compatible with the presence of two monomers in the asymmetric unit, with a corresponding Matthews coefficient VM of 2.52 Å(3) Da(-1) and a solvent content of 51.2%. The crystal structure of MDH has been solved by molecular replacement and is currently under refinement.

Expression and identification of a thermostable malate dehydrogenase from multicellular prokaryote Streptomyces avermitilis MA-4680.

A malate dehydrogenase (MDH) from Streptomyces avermitilis MA-4680 (SaMDH) has been expressed and purified as a fusion protein. The molecular mass of SaMDH is about 35 kDa determined by SDS-PAGE. The recombinant SaMDH has a maximum activity at pH 8.0. The enzyme shows the optimal temperature around 42 °C and displays a half-life (t(1/2)) of 160 min at 50°C which is more thermostable than reported MDHs from most bacteria and fungi. The k(cat) value of SaMDH is about 240-fold of that for malate oxidation. In addition, the k(cat)/K(m) ratio shows that SaMDH has about 1,246-fold preference for oxaloacetate (OAA) reduction over L-malate oxidation. The recombinant SaMDH may also use NADPH as a cofactor although it is a highly NAD(H)-specific enzyme. There was no activity detected when malate and NADP(+) were used as substrates. Substrate inhibition studies show that SaMDH activity is strongly inhibited by excess OAA with NADH, but is not sensitive to excess L-malate. Enzymatic activity is enhanced by the addition of Na(+), NH(4)(+), Ca(2+), Cu(2+) and Mg(2+) and inhibited by addition of Hg(2+) and Zn(2+). MDH is widely used in coenzyme regeneration, antigen immunoassays and bioreactors. The enzymatic analysis could provide the important basic knowledge for its utilizations.

Cloning, sequencing and functional expression of cytosolic malate dehydrogenase from Taenia solium: Purification and characterization of the recombinant enzyme.

We report herein the complete coding sequence of a Taenia solium cytosolic malate dehydrogenase (TscMDH). The cDNA fragment, identified from the T. solium genome project database, encodes a protein of 332 amino acid residues with an estimated molecular weight of 36517Da. For recombinant expression, the full length coding sequence was cloned into pET23a. After successful expression and enzyme purification, isoelectrofocusing gel electrophoresis allowed to confirm the calculated pI value at 8.1, as deduced from the amino acid sequence. The recombinant protein (r-TscMDH) showed MDH activity of 409U/mg in the reduction of oxaloacetate, with neither lactate dehydrogenase activity nor NADPH selectivity. Optimum pH for enzyme activity was 7.6 for oxaloacetate reduction and 9.6 for malate oxidation. K(cat) values for oxaloacetate, malate, NAD, and NADH were 665, 47, 385, and 962s(-1), respectively. Additionally, a partial characterization of TsMDH gene structure after analysis of a 1.56Kb genomic contig assembly is also reported.

[Mechanism of malate dehydrogenase isoform formation in Sphaerotilus natans D-507 under different cultivation conditions].

Electrophoretically homogenous preparations of malate dehydrogenase (MDH) isoforms of the bacteria Sphaerotilus natans D-507 with specific activity 7.46 U/mg and 5.74 U/mg with respect to protein concentration have been obtained. The dimeric isoform of the enzyme was shown to function under organotrophic growth conditions, whereas the tetrameric isoform was induced under mixotrophic cultivation conditions. PCR-analysis revealed a single gene encoding the malate dehydrogenase molecule. The topography of the MDH isoform surface was studied by atomic-force microscopy, and a 3D-structure of the enzyme was obtained. Spectraphotometric analysis data allowed us to suggest that stabilization of the tetrameric form of MDH is due to additional bounds implicated in the quaternary structure formation.

Structural analysis and reaction mechanism of malate dehydrogenase from Geobacillus stearothermophilus.

Malate dehydrogenase (MDH) catalyzes the reversible reduction of oxaloacetate (OAA) to L-malate using nicotinamide adenine dinucleotide hydrogen. MDH has two characteristic loops, the mobile loop and the catalytic loop, in the active site. On binding to the substrate, the enzyme undergoes a structural change from the open-form, with an open conformation of the mobile loop, to the closed-form, with the loop in a closed conformation. In this study, three crystals of MDH from a moderate thermophile, Geobacillus stearothermophilus (gs-MDH) were used to determine four different enzyme structures (resolutions, 1.95-2.20 Å), each of which was correspondingly assigned to its four catalytic states. Two OAA-unbound structures exhibited the open-form, while the other two OAA-bound structures exhibited both the open- and closed-form. The structural analysis suggested that the binding of OAA to the open-form gs-MDH promotes conformational change in the mobile loop and simultaneously activates the catalytic loop. The mutations on the key amino acid residues involving the proposed catalytic mechanism significantly affected the gs-MDH activity, supporting our hypothesis. These findings contribute to the elucidation of the detailed molecular mechanism underlying the substrate recognition and structural switching during the MDH catalytic cycle.

Isolation and properties of malic enzyme and its gene in Rhodopseudomonas palustris No. 7.

Malic enzyme (ME) was purified as an electrophoretically homogenous protein from Rhodopseudomonas palustris No. 7. The molecular weight of ME was estimated to be 650 kDa and that of its subunit, 86 kDa. ME activity was remarkably enhanced by di- and mono-valent cations, and the K(a) values for Mg(2+) and NH(4)(+) were 0.26 and 0.56 mM respectively. Purified ME used both NAD(+) and NADP(+) as electron acceptors, with K(m) values of 0.11 and 1.8 mM. The K(m) value for L-malate was 1.7 mM using NAD(+) as electron acceptor. Gene cloning of the ME indicated that the ME from R. palustris strain No. 7 was composed of 774 amino acids encompassing the ME and phosphotransacetylase domains, although purified ME displayed no phosphotransacetylase activity. ME activity was inhibited by acetyl-CoA, oxaloacetate, and fructose-6-phosphate. These results suggest that ME plays an important role in the metabolic regulation of R. palustris No. 7 under photoheterotrophic conditions.

A dye-affinity cryogel membrane for malate dehydrogenase purification from Saccharomyces cerevisiae.

Cibacron blue F3GA functionalized poly(hydroxyethyl methacrylate) cryogel membranes were prepared and applied for a simple purification of malate dehydrogenase (MDH) from crude extract of Saccharomyces cerevisiae. Swelling tests, scanning electron microscopy, surface area measurements and Fourier transform infrared (FTIR) spectroscopy techniques were used for the characterization of dye-affinity cryogel membranes. Following cell homogenization and extraction, MDH was purified using the dye-affinity cryogel membranes at a high yield of 80.5% with 54-fold purification. Maximum MDH adsorption amount was determined to be 267.7 mg/g of membranes at pH 7.4, 25 °C and a flow rate of 1.0 mL/min. Interestingly, the cryogel membranes were used for several purification runs without any significant decrease in MDH adsorption capacity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was carried out to assess the purity of the eluted MDH. The obtained results highlight the dye-affinity cryogel membranes as powerful dye affinity adsorbents for MDH purification from S. cerevisiae.

Identification of major allergens in watermelon.

BACKGROUND: Watermelon is a worldwide consumed Cucurbitaceae fruit that can elicit allergic reactions. However, the major allergens of watermelon are not known. The aim of this study is to identify and characterize major allergens in watermelon. METHODS: Twenty-three patients allergic to watermelon took part in the study. The diagnosis was based on a history of symptoms and positive skin prick-prick tests to watermelon, confirmed by positive open oral challenge testing to watermelon pulp. Allergenic components were detected by SDS-PAGE and immunoblotting. Molecular characterization of IgE-binding bands was performed by N-terminal amino acid sequencing and mass spectrometry. Allergens were purified combining several chromatographic steps. RESULTS: Several IgE binding bands (8-120 kDa) were detected in watermelon extract. Three major allergens were identified as malate dehydrogenase (36 kDa), triose phosphate isomerase (28 kDa) and profilin (13 kDa). Purified allergens individually inhibited IgE binding to the whole watermelon extract. CONCLUSIONS: All in all these results indicate that malate dehydrogenase, triose phosphate isomerase and profilin are major allergens involved in watermelon allergy.

Respiration and protein synthesis in Escherichia coli membrane-envelope fragments. VI. Solubilization and characterization of the electron transport chain.

Membranes obtained from Escherichia coli have been solubilized with deoxycholate. The solubilized dehydrogenases and cytochromes are not sedimented at 105,000 g. These components readily penetrate the "included space" of Sepharose 4B (Pharmacia Fine Chemicals Inc., Uppsala, Sweden) and polyacrylamide gels and have been fractionated on the basis of molecular size. Solubilization destroys nicotinamide adenine dinucleotide, reduced form (NADH) oxidase and D-lactate oxidase activities, but leaves an appreciable part of the original succinoxidase activity intact. Evidence for a succinate dehydrogenase-cytochrome b(1) complex is given. Menadione added to the solubilized preparation does not elicit NADH oxidase activity nor stimulate the existing succinoxidase activity, but does provoke an active D-lactate oxidase activity. This D-lactate oxidase activity, however, does not use cytochromes and is not sensitive to cyanide.