Amino acid sequence homology among the 2-hydroxy acid dehydrogenases: mitochondrial and cytoplasmic malate dehydrogenases form a homologous system with lactate dehydrogenase.

Birktoft, Jens J.; Fernley, Ross T.; Bradshaw, Ralph A.; Banaszak, Leonard J.

doi:10.1073/pnas.79.20.6166

Cited by 123 publications

(88 citation statements)

References 27 publications

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“…The plMDH forms also contain a group of conserved residues (IWGNH) near the active site histidine (Metzler et al, 1989). This same motif can be found in lactate dehydrogenases and cMDH forms from alfalfa and mouse (Birktoft et al, 1982;Metzler et al, 1989). The maize plMDH also contains a consensus sequence of CFGVFC that has been shown to be the site of thioredoxin regulation of the isoform (Hatch and Slack, 1969).…”

Section: Discussionmentioning

confidence: 82%

“…Several MDH cDNA clones have been isolated and sequences compared from both plant (Cretin et al, 1988;Gietl, 1992;Guex et al, 1995;Kim and Smith, 1994;Metzler et al, 1989) and animal sources (Birktoft et al, 1982;Grant et al, 1986;Joh et al, 1987a;Joh et al, 1987b). The sequences of both the mouse and yeast c-and mMDH genes have been reported (Minard and McAlister-Henn, 1991;Setoyama et al, 1988;Takeshima et al, 1988;Thompson et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

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Alfalfa malate dehydrogenase (MDH): molecular cloning and characterization of five different forms reveals a unique nodule‐enhanced MDH

Miller¹,

Driscoll²,

Gregerson³

et al. 1998

The Plant Journal

151

100

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SummaryMalate dehydrogenase (MDH) catalyzes the readily reversible reaction of oxaloacetate ≤ malate using either NADH or NADPH as a reductant. In plants, the enzyme is important in providing malate for C 4 metabolism, pH balance, stomatal and pulvinal movement, respiration, β-oxidation of fatty acids, and legume root nodule functioning. Due to its diverse roles the enzyme occurs as numerous isozymes in various organelles. While antibodies have been produced and cDNAs characterized for plant mitochondrial, glyoxysomal, and chloroplast forms of MDH, little is known of other forms. Here we report the cloning and characterization of cDNAs encoding five different forms of alfalfa MDH, including a plant cytosolic MDH (cMDH) and a unique novel nodule-enhanced MDH (neMDH). Phylogenetic analyses show that neMDH is related to mitochondrial and glyoxysomal MDHs, but diverge from these forms early in land plant evolution. Four of the five forms could effectively complement an E. coli Mdh -mutant. RNA and protein blots show that neMDH is most highly expressed in effective root nodules. Immunoprecipitation experiments show that antibodies produced to cMDH and neMDH are immunologically distinct and that the neMDH form comprises the major form of total MDH activity and protein in root nodules. Kinetic analysis showed that neMDH has a turnover rate and specificity constant that can account for the extraordinarily high synthesis of malate in nodules.

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Section: Discussionmentioning

confidence: 82%

Section: Introductionmentioning

confidence: 99%

Alfalfa malate dehydrogenase (MDH): molecular cloning and characterization of five different forms reveals a unique nodule‐enhanced MDH

Miller¹,

Driscoll²,

Gregerson³

et al. 1998

The Plant Journal

151

100

View full text Add to dashboard Cite

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“…1) for mMDH-FITC conjugates prepared using protocol P, a Po value for fluorescein of 0.47 (Hamman et al, 1996) and the observed lifetime of 4.16 ns, the calculated rotational relaxation time calculated is 25 ns. The Debye rotational relaxation time expected for a rigid, spherical 68 kDa protein, in aqueous buffer at 20 "C (77 = 1 .OO cp), assuming a partial specific volume of 0.75 mL/g [calculated from the amino acid sequence (Birktoft et al, 1982) using the method of Cohn and Edsall (1943)] and a hydration of 0.22 mL/g, is 74 ns. Clearly, the experimentally determined rotational relaxation time is significantly shorter than that expected for a 68 kDa spherical protein, and we may presume that the probe is monitoring more than one rotational motion.…”

Section: Discussionmentioning

confidence: 99%

Aggregation states of mitochondrial malate dehydrogenase

et al. 1998

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The oligomeric state of fluorescein-labeled mitochondrial malate dehydrogenase (L-malate NAD+ oxidoreductase; mMDH; EC 1.1.1.37), as a function of protein concentration, has been examined using steady-state and dynamic polarization methodologies. A "global" rotational relaxation time of 103 k 7 ns was found for micromolar concentrations of mMDH-fluorescein, which is consistent with the reported size and shape of mMDH. Dilution of the mMDHfluorescein conjugates, prepared using a phosphate buffer protocol, to nanomolar concentrations had no significant effect on the rotational relaxation time of the adduct, indicating that the dimer-monomer dissociation constant for mMDH is below M. In contrast to reports in the literature suggesting a pH-dependent dissociation of mMDH, the oligomeric state of this mMDH-fluorescein preparation remained unchanged between pH 5.0 and 8.0. Application of hydrostatic pressure up to 2.5 kilobars was ineffective in dissociating the mMDH dimer. However, the mMDH dimer was completely dissociated in 1.5 M guanidinium hydrochloride. Dilution of a mMDH-fluorescein conjugate, prepared using a Tris buffer protocol, did show dissociation, which can be attributed to aggregates present in these preparations. These results are considered in light of the disparities in the literature concerning the properties of the mMDH dimer-monomer equilibrium.Keywords: dissociation; fluorescence polarization; mitochondrial malate dehydrogenase: time resolved Porcine heart mitochondrial malate dehydrogenase (mMDH) [(S)-malate:NAD+ oxidoreductase, E.C. 1 .I. 1.371 is a dimeric enzyme composed of identical subunits, each with 314 amino acids, which catalyzes the interconversion of L-malate and oxaloacetate utilizing NAD+ as a coenzyme. In 1970s and 1980s, several groups studied the dimer-monomer equilibrium of mMDH using different approaches. Shore and Chakrabarti (1976) reported fluorescence polarization studies on enzyme labeled with either FITC or fluorescamine and, in both cases, reported finding a concentrationdependent dissociation with a dissociation constant (&) equal to 2 X IOu7 M at 23 "C in pH 8.0, 50 mM Tris-acetate buffer. Bleile et al. (1977) and Hodges et al. (1977) reported gel filtration chromatography and sedimentation velocity ultracentrifugation studies indicating dissociation constants for the dimer similar to those reported by Shore and Chakrabarti. Frieden et al. (1978), however, reported enzyme kinetic studies suggesting that mMDH did not undergo dissociation even at 10" M. Jaenicke et al. (1979), using gel filtration chromatography, also concluded that mMDH remained a dimer over the concentration range of 1.67 X IO-' M to 2.9 X M in 0.2 M phosphate buffer, pH 7.6, 20°C. Wood et al. (1978Wood et al. ( , 1981 and Hodges et al. (1977) reported a pH dependence of the dimer/monomer equilibrium, suggesting that pH values below 7 promote dissociation; specifically, a dissociation constant greater than 2 X lop4 M was reported at pH 5.0, while at pH 7.5 this value was given as less than M (...

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“…Investigation of the optimum pH for m-MDH activity in the oxaloacetate reductase direction was performed with 200 lM oxaloacetate and 200 lM NADH over a wide pH range (4)(5)(6)(7)(8)(9)(10)(11)(12) and at 30°C. Determination of the pH optimum in the malate oxidation direction was achieved with 10 mM malate and 1 mM NAD + as substrate and cosubstrate, respectively, over the same pH range at 30°C.…”

Section: Determination Of Selected Biochemical Propertiesmentioning

confidence: 99%

Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii

Maloney

Callan

Murray

et al. 2004

European Journal of Biochemistry

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Mitochondrial malate dehydrogenase (m-MDH; EC 1.1.1.37), from mycelial extracts of the thermophilic, aerobic fungus Talaromyces emersonii, was purified to homogeneity by sequential hydrophobic interaction and biospecific affinity chromatography steps. Native m-MDH was a dimer with an apparent monomer mass of 35 kDa and was most active at pH 7.5 and 52°C in the oxaloacetate reductase direction. Substrate specificity and kinetic studies demonstrated the strict specificity of this enzyme, and its closer similarity to vertebrate m-MDHs than homologs from invertebrate or mesophilic fungal sources. The fulllength m-MDH gene and its corresponding cDNA were cloned using degenerate primers derived from the N-terminal amino acid sequence of the native protein and multiple sequence alignments from conserved regions of other m-MDH genes. The m-MDH gene is the first oxidoreductase gene cloned from T. emersonii and is the first full-length m-MDH gene isolated from a filamentous fungal species and a thermophilic eukaryote. Recombinant m-MDH was expressed in Escherichia coli, as a His-tagged protein and was purified to apparent homogeneity by metal chelate chromatography on an Ni 2+ -nitrilotriacetic acid matrix, at a yield of 250 mg pure protein per liter of culture. The recombinant enzyme behaved as a dimer under nondenaturing conditions. Expression of the recombinant protein was confirmed by Western blot analysis using an antibody against the His-tag. Thermal stability studies were performed with the recombinant protein to investigate if results were consistent with those obtained for the native enzyme.Keywords: malate dehydrogenase; filamentous fungus; bio-specific affinity chromatography; thermophilic eukaryote; Talaromyces emersonii.Malate dehydrogenase (MDH) catalyzes the pyridine nucleotide-dependent interconversion of malate and oxaloacetate in the tricarboxylic acid cycle. It is also thought to have a role in the malate/aspartate shuttle across the inner mitochondrial membrane. In most eukaryotic cells there are two major isoenzymes of MDH, cytosolic MDH (c-MDH) and mitochondrial MDH (m-MDH) [1].The majority of m-MDHs isolated to date from eukaryotic sources are homodimers of identical subunits, with a monomer size of 34 kDa. Mitochondrial MDH displays a complex regulatory pattern that involves allosteric activation [2], membrane interaction [3] and formation of multienzyme complexes [4] with enzymes such as citrate synthase, aspartate aminotransferase and other mitochondrial components [5][6][7][8][9][10]. Much of the previous research has focused on the comparison of primary and three-dimensional structures of mitochondrial and cytoplasmic forms of MDH isolated from a single source, e.g. pig heart tissue [11,12]. In contrast to bacterial, vertebrate and invertebrate m-MDH, a paucity of information exists on fungal m-MDH with significantly more known about yeast m-MDH than homologs from filamentous fungal species. Previous research by Dalhaus et al. [13] and Goward and Nicholls [14] has proposed that investigation...

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Amino acid sequence homology among the 2-hydroxy acid dehydrogenases: mitochondrial and cytoplasmic malate dehydrogenases form a homologous system with lactate dehydrogenase.

Cited by 123 publications

References 27 publications

Alfalfa malate dehydrogenase (MDH): molecular cloning and characterization of five different forms reveals a unique nodule‐enhanced MDH

Alfalfa malate dehydrogenase (MDH): molecular cloning and characterization of five different forms reveals a unique nodule‐enhanced MDH

Aggregation states of mitochondrial malate dehydrogenase

Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii

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