Metaphosphate: A new phosphoryl donor for NAD phosphorylation.

Murata, Kousaku; Uchida, Tomofumi; Tani, Keiko; Chibata, Ichiro

doi:10.1271/bbb1961.44.61

Cited by 22 publications

(15 citation statements)

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“…NAD kinase was assayed by means of a two‐step method as described previously [10] in a reaction mixture (1.0 mL) consisting of 5.0 m m NAD, 5.0 m m MgCl 2 , 100 m m Tris/HCl pH 7.0, and 5.0 m m ATP. NADP was determined enzymatically with isocitrate dehydrogenase [11].…”

Section: Methodsmentioning

confidence: 99%

Molecular characterization of Escherichia coli NAD kinase

Kawai

Mori

Mukai

et al. 2001

European Journal of Biochemistry

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NAD kinase was purified to homogeneity from Escherichia coli MG1655. The enzyme was a hexamer consisting of 30 kDa subunits and utilized ATP or other nucleoside triphosphates as phosphoryl donors for the phosphorylation of NAD, most efficiently at pH 7.5 and 60 8C. The enzyme could not use inorganic polyphosphates as phosphoryl donors and was designated as ATP±NAD kinase. The N-terminal amino-acid sequence of the purified enzyme was encoded by yfjB, which had been deposited as a gene of unknown function in the E. coli whole genomic DNA sequence database. yfjB was cloned and expressed in E. coli BL21(DE3)pLysS. The purified product (YfjB) showed NAD kinase activity, and was identical to ATP±NAD kinase purified from E. coli MG1655 in molecular structure and other enzymatic properties. ) have been purified to homogeneity. Among them, the enzymes of M. flavus and M. tuberculosis have been shown to utilize inorganic polyphosphate [poly(P)] in addition to ATP, and were designated as poly(P)/ATP±NAD kinase' [7]. However, the gene for NAD kinase, to the best of our knowledge, had never been cloned from any organism before we isolated the gene for the enzyme from M. tuberculosis [7].The lack of information about the NAD kinase gene has hindered definative understanding of NADP metabolism in organisms. To overcome this, and to obtain new insights regarding the regulation of the cellular redox system, we have attempted to isolate the NAD kinase gene of Escherichia coli. NAD kinase has been partially purified from E. coli and some properties of the enzyme have been preliminarily reported [8]. In this study, we identified the gene for NAD kinase of E. coli through purification and analysis of the primary structure of the enzyme, and revealed that the E. coli NAD kinase can utilize ATP and other nucleoside triphosphates, but not poly(P), and designated the enzyme as ATP±NAD kinase to distinguish it from poly(P)/ATP±NAD kinase of M. flavus and M. tuberculosis. M A T E R I A L S A N D M E T H O D SBacterial strains E. coli MG1655 was cultured at 37 8C in YGD medium comprising 0.1% (NH4) 2 SO4, 0.05% MgSO 4 7H 2 O, 0.1% KH 2 PO4, 0.4% Na 2 HPO 4 , 0.5% yeast extract, and 0.5% glucose (pH 7.2). As a host for plasmid amplification, E. coli DH5a (Toyobo, Osaka, Japan) was routinely cultured at 37 8C in Luria±Bertani medium [9] supplemented with ampicillin (100 mg´mL 21 ). The growth conditions for the derivative strains of E. coli BL21(DE3) pLysS (Novagen, Darmstadt, Germany) are described below.Assays NAD kinase was assayed by means of a two-step method as described previously [10] in a reaction mixture (1.0 mL) consisting of 5.0 mm NAD, 5.0 mm MgCl 2 , 100 mm Tris/ HCl pH 7.0, and 5.0 mm ATP. NADP was determined enzymatically with isocitrate dehydrogenase [11]. One unit of enzyme activity was defined as 1.0 nmol of NADP produced in 1 min at 37 8C, and specific activity was expressed in U´mg protein 21 . K m , V max and Hill coefficients (h) were determined by means of Lineweaver±Burk and Hill plots, respectively. The rate constan...

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Section: Methodsmentioning

confidence: 99%

Molecular characterization of Escherichia coli NAD kinase

Kawai

Mori

Mukai

et al. 2001

European Journal of Biochemistry

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121

123

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“…This enzyme was able to degrade synthetic polyphosphate with a chain length of 15 phospho-groups. In Escherichia coli polyphosphate kinase was found (13), whereas Mycobacterium phlei contained polyphosphate glucokinase (23), and polyphosphate-dependent NAD-kinase was detected in Acetobacter, Achromobacter, Brevibacterium, Corynebacterium, and Micrococcus species (18). Activities of the last three enzymes were absent in cell extracts of Acinetobacter strain 210A (26).…”

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confidence: 98%

Polyphosphate-degrading enzymes in Acinetobacter spp. and activated sludge

Groenestijn

Bentvelsen

Deinema

et al. 1989

Appl Environ Microbiol

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Polyphosphate-degrading enzymes were studied in Acinetobacter spp. and activated sludge. Polyphosphate: AMP phosphotransferase activity in Acinetobacter strain 210A decreased with increasing growth rates. The activity of this enzyme in cell extracts of Acinetobacter strain 210A was maximal at a pH of 8.5 and a temperature of 40°C and was stimulated by (NH4)2SO4. The Km for AMP was 0.6 mM, and the Vmax was 60 nmol/min per mg of protein. Cell extracts of this strain also contained polyphosphatase, which was able to degrade native polyphosphate and synthetic magnesium polyphosphate and was strongly stimulated by 300 to 400 mM NH4Cl. A positive correlation was found between polyphosphate:AMP phosphotransferase activity, adenylate kinase activity, and phosphorus accumulation in six Acinetobacter strains. Significant activities of polyphosphate kinase were detected only in strain P, which contained no polyphosphate:AMP phosphotransferase. In samples of activated sludge from different plants, the activity of adenylate kinase correlated well with the ability of the sludge to remove phosphate biologically from wastewater.

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“…Instead of authen tic poly(P) or ATP, the 32 Plabeled poly(P) was used as a phosphoryl donor in the reaction mixture (20 ml) for the assaying of NAD kinase, as described above. After the addition of the puried reMfnkC (0.10 mg) to the mixture, the reaction mixture was incubated at 379 C. At the prescribed times, 1.0 ml of the reaction mixture was withdrawn, spotted onto a silica gel, and then developed with a solvent system [isobutyrate500 mM NH 4 OH (5: 12) As controls, authentic ATP, ADP, NAD, NADP, Pi, and poly(P) (metaphosphate) were also developed similarly. The developed 32 Plabeled compounds were visualized with an Imaging plate type BASIII (Fuji Photo Film, Tokyo, Japan), and the nucleo tides with exposure to UV light.…”

Section: Methodsmentioning

confidence: 99%

Primary Structure of Inorganic Polyphosphate/ATP-NAD Kinase fromMicrococcus flavus, and Occurrence of Substrate Inorganic Polyphosphate for the Enzyme

Kawai¹,

Mori²,

Murata³

2003

Bioscience, Biotechnology, and Biochemistry

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The gene encoding an inorganic polyphosphate/ATP-NAD kinase was cloned from Micrococcus flavus, and its primary structure was analyzed. Alignment of the primary structure with those of other characterized NAD kinases revealed candidate amino acid residues, mainly charged ones, that would be related to inorganic polyphosphate use. The alignment also showed that the primary structure found carried a protruding C-terminal polypeptide. Although the C-terminal polypeptide was demonstrated to be dispensable for the kinase activities, and was proposed to be removed in M. flavus, the entire primary structure including the C-terminal polypeptide was homologous with that of the ATP synthase beta chain. The inorganic polyphosphate used by the inorganic polyphosphate/ATP-NAD kinase as a phosphoryl donor was isolated from cells of M. flavus, suggesting that the ability of the enzyme to use inorganic polyphosphate is of physiological significance and is not an evolutionary trait alone.

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Metaphosphate: A new phosphoryl donor for NAD phosphorylation.

Cited by 22 publications

References 1 publication

Molecular characterization of Escherichia coli NAD kinase

Molecular characterization of Escherichia coli NAD kinase

Polyphosphate-degrading enzymes in Acinetobacter spp. and activated sludge

Primary Structure of Inorganic Polyphosphate/ATP-NAD Kinase fromMicrococcus flavus, and Occurrence of Substrate Inorganic Polyphosphate for the Enzyme

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