Purification and Kinetic Characterization ofHaemophilus parasuisMalate Dehydrogenase

Wise, Darla J.; Anderson, Christopher R.; Anderson, Bruce M.

doi:10.1006/abbi.1997.0186

Cited by 15 publications

(10 citation statements)

References 21 publications

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“…Many NAD-dependent dehydrogenases follow a steady state ordered mechanism in which NAD is the first substrate to bind (47,48 acid-specific family, the kinetic mechanism has been determined for only a single member. This enzyme, 2-hydroxyisocaproate dehydrogenase, follows the rapid equilibrium ordered mechanism with NAD binding first in the forward direction but follows the steady state ordered mechanism with NADH binding first in the reverse direction (49).…”

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confidence: 99%

Purification and Characterization of a Novel Phosphorus-oxidizing Enzyme from Pseudomonas stutzeri WM88

Costas¹,

White²,

Metcalf

2001

Journal of Biological Chemistry

154

155

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The ptxD gene from Pseudomonas stutzeri WM88 encoding the novel phosphorus oxidizing enzyme NAD:phosphite oxidoreductase (trivial name phosphite dehydrogenase, PtxD) was cloned into an expression vector and overproduced in Escherichia coli. The heterologously produced enzyme is indistinguishable from the native enzyme based on mass spectrometry, amino-terminal sequencing, and specific activity analyses. Recombinant PtxD was purified to homogeneity via a two-step affinity protocol and characterized. The enzyme stoichiometrically produces NADH and phosphate from NAD and phosphite. The reverse reaction was not observed. Gel filtration analysis of the purified protein is consistent with PtxD acting as a homodimer. PtxD has a high affinity for its substrates with K m values of 53.1 ؎ 6.7 M and 54.6 ؎ 6.7 M, for phosphite and NAD, respectively. V max and k cat were determined to be 12.2 ؎ 0.3 mol min ؊1 mg ؊1 and 440 min ؊1. NADP can substitute poorly for NAD; however, none of the numerous compounds examined were able to substitute for phosphite. Initial rate studies in the absence or presence of products and in the presence of the dead end inhibitor sulfite are most consistent with a sequential ordered mechanism for the PtxD reaction, with NAD binding first and NADH being released last. Amino acid sequence comparisons place PtxD as a new member of the D-2-hydroxyacid NAD-dependent dehydrogenases, the only one to have an inorganic substrate. To our knowledge, this is the first detailed biochemical study on an enzyme capable of direct oxidation of a reduced phosphorus compound.Phosphorus is widely reported to be a redox conservative element in biological systems, with the sum total of phosphorus biochemistry consisting of the formation and hydrolysis of phosphate-ester bonds. These reports imply that reduced phosphorus compounds are not important in living systems and that enzymatically catalyzed redox reactions of phosphorus compounds do not occur; however, an increasing body of evidence indicates that this is not the case. Although it is true that inorganic phosphate (P valence ϩ5) is the principal form of phosphorus in living systems and that phosphate-esters play a critical role in phosphate biochemistry, it is now clear that reduced phosphorus compounds of both natural and xenobiotic origin play important roles in numerous biological systems. Accordingly, many organisms have been shown to possess metabolic pathways for reduction of phosphate to a variety of reduced phosphorus compounds (1-3); others have been shown to possess metabolic pathways for oxidation of reduced phosphorus compounds (4 -9). Among the most striking of these is a recently isolated sulfate-reducing bacterium that obtains all of the energy it requires for growth from the oxidation of phosphite (ϩ3 valence) to phosphate (10).Unfortunately, detailed studies examining the mechanisms of biological phosphorus oxidation and reduction are scarce. This is particularly true with regard to the biochemical characterization of enzymes involved in reduced p...

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confidence: 99%

Purification and Characterization of a Novel Phosphorus-oxidizing Enzyme from Pseudomonas stutzeri WM88

Costas¹,

White²,

Metcalf

2001

Journal of Biological Chemistry

154

155

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“…AMP shows higher inhibition to CsmMDH than to Cs-cMDH, although it did not show very high inhibitory effect to both MDHs. Wise et al (1997) found that although AMP is not a strong inhibitor of mMDH of Haemophilus parasuis, it inhibits the growth of H. parasuis more effectively than ADP-ribose (a stronger inhibitor of mMDH). Whether AMP can be a helminthic to liver fluke or can inhibit the growth of liver fluke requires further tests.…”

Section: Discussionmentioning

confidence: 96%

“…3), the recombinant Cs-cMDH and the recombinant Cs-mMDH cleaved by thrombin all settled in the position of approximately 36 kDa. In all cases, the MDH was a homodimer or a homotetramer with a subunit molecular mass of 30-38 kDa (van Kuijk and Stams 1996;Wise et al 1997;Kirby 2000).…”

Section: Discussionmentioning

confidence: 98%

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Enzymatic and physico-chemical characteristics of recombinant cMDH and mMDH of Clonorchis sinensis

Zheng¹,

Huang²,

et al. 2006

Parasitol Res

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The cytosol and mitochondrial malate dehydrogenases (MDHs, EC 1.1.1.37) of Clonorchis sinensis were expressed in Escherichia coli as a fusion protein with a 6xHis and GST tag, respectively. The cytosol MDH of Clonorchis sinensis (Cs-cMDH) has higher resistibility to acid than mitochondrial MDH (Cs-mMDH). The Cs-cMDH also has higher heat resistibility and thermal stability than Cs-mMDH. Although there is only 22.8% identity between the amino acid sequences of Cs-cMDH and Cs-mMDH, they share several conserved residues. There are some differences between the circular dichroism spectra of Cs-cMDH and Cs-mMDH, but they have approximate percentages of helix. 4,4'-Bisdimethylamino diphenylcarbinol can decrease the Cs-mMDH activity but not the Cs-cMDH activity. Paraziquantel, metronidazole and albendazole did not inhibit the enzymes' activity, but adenosine 5'-monophosphate showed competitive inhibition to enzyme, with the Ki for Cs-cMDH and Cs-mMDH being 2.81 and 0.49 mM, respectively.

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“…Enzymatic assay of mMDH Enzymatic assay of mMDH was performed at 25°C according the method of Wise et al (1997). In each case, the change in absorbance at 340 nm because of the oxidation or reduction of NADH was measured on UV-260 recording spectrophotometer (Shimadzu, Kyoto, Japan).…”

Section: Sds-page and Western-blot Analysismentioning

confidence: 99%

Cloning and expression of mitochondrial malate dehydrogenase of Clonorchis sinensis

Zheng¹,

Huang²,

et al. 2008

Parasitol Res

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The NAD-dependent mitochondrial malate dehydrogenase (mMDH, EC1.1.1.37) plays pivotal roles in tricarboxylic acid and is crucial for the survival and pathogenecity of parasites. A cDNA, which was identified by high throughput sequencing from the cDNA library constructed from adult Clonorchis sinensis, encoded a putative peptide of 341 amino acids with more than 50% identity with mMDHs from other organisms. The mMDH was expressed in Escherichia coli as the recombinant protein with a GST tag and purified by glutathione-Sepharose 4B column. The recombinant mMDH showed MDH activity of 63.6 U/mg, without lactate dehydrogenase activity and NADPH selectivity. The kinetic constants of recombinant mMDH were determined.

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Purification and Kinetic Characterization ofHaemophilus parasuisMalate Dehydrogenase

Cited by 15 publications

References 21 publications

Purification and Characterization of a Novel Phosphorus-oxidizing Enzyme from Pseudomonas stutzeri WM88

Purification and Characterization of a Novel Phosphorus-oxidizing Enzyme from Pseudomonas stutzeri WM88

Enzymatic and physico-chemical characteristics of recombinant cMDH and mMDH of Clonorchis sinensis

Cloning and expression of mitochondrial malate dehydrogenase of Clonorchis sinensis

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