Primary Structure of Inorganic Polyphosphate/ATP-NAD Kinase from<i>Micrococcus flavus</i>, and Occurrence of Substrate Inorganic Polyphosphate for the Enzyme

Kawai, Shigeyuki; Mori, Shiro; Murata, Kousaku

doi:10.1271/bbb.67.1751

Cited by 15 publications

(20 citation statements)

References 14 publications

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“…Purifications of Mfnk, Ppnk, and YfjB-The Mfnk of M. flavus, Ppnk of M. tuberculosis H37Rv, and YfjB of E. coli were expressed in E. coli BL21(DE3)pLysS (Novagen, Darmstadt, Germany) and purified to homogeneity, which was judged by SDS-PAGE (15), as described previously (6,11,16). The purified enzymes were dialyzed overnight against 10 mM potassium phosphate (pH 7.0) at 4°C and used for the assays.…”

Section: Nadmentioning

confidence: 99%

“…The mutagenized plasmid encoding the NadK mutant NadK R180G, in which Arg-180 is converted to Gly, was constructed using pMK225 (pET-17b (Novagen) containing the NadK gene) (8) as the template. The mutagenized plasmid encoding the Mfnk mutant (namely Mfnk G183R, in which Gly-183 is converted to Arg) was constructed using pSK177 (pET-17b containing the Mfnk gene) (16) as the template. The mutagenized plasmids thus constructed were confirmed by DNA sequencing and introduced into E. coli BL21(DE3)pLysS.…”

Section: Nadmentioning

confidence: 99%

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Molecular Conversion of NAD Kinase to NADH Kinase through Single Amino Acid Residue Substitution

Mori

Kawai

Shi

et al. 2005

Journal of Biological Chemistry

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NAD kinase phosphorylates NAD؉ to form NADP ؉ and is strictly specific to NAD ؉ , whereas NADH kinase phosphorylates both NAD ؉ and NADH, thereby showing relaxed substrate specificity. Based on their primary and tertiary structures, the difference in the substrate specificities between NAD and NADH kinases was proposed to be caused by one aligned residue: Gly or polar amino acid (Gln or Thr) in five NADH kinases and a charged amino acid (Arg) in two NAD kinases. The substitution of Arg with Gly in the two NAD kinases relaxed the substrate specificity (i.e. converted the NAD kinases to NADH kinases). The substitution of Arg in one NAD kinase with polar amino acids also relaxed the substrate specificity, whereas substitution with charged and hydrophobic amino acids did not show a similar result. In contrast, the substitution of Gly with Arg in one NADH kinase failed to convert it to NAD kinase. These results suggest that a charged or hydrophobic amino acid residue in the position of interest is crucial for strict specificity of NAD kinases to NAD ؉ , whereas Gly or polar amino acid residue is not the sole determinant for the relaxed substrate specificity of NADH kinases. The significance of the conservation of the residue at the position in 207 NAD kinase homologues is also discussed. NADϩ and NADH are almost exclusively involved in catabolic reactions, whereas NADP ϩ and NADPH primarily participate in anabolic reactions and cellular defense against oxidative stress (1, 2). A recent study has revealed that in addition to these functions, NAD ϩ is also used as a substrate for mono-and poly-ADP ribosylations and for the formation of cyclic ADPribose, dimeric ADP-ribose, and nicotinic acid adenine nucleotide (3). Thus, the regulation of the cellular concentrations of NAD ϩ , NADP ϩ , and their respective reduced equivalents (NADH and NADPH) is considered to govern numerous cellular biological processes.NAD kinase (EC 2.7.1.23) catalyzes the formation of NADP ϩ through the phosphorylation of NAD ϩ and is a key enzyme required for the regulation of cellular concentrations of NAD ϩ and NADP ϩ (4). Based on the phosphoryl donor specificity, NAD kinase is divided into two types: inorganic polyphosphate (poly(P)) 1 /ATP-NAD kinase and ATP-NAD kinase (5, 6). Poly(P) is a linear polymer of orthophosphate residues linked by phosphoanhydride bonds (7). Poly(P)/ATP-NAD kinase utilizes both poly(P) and ATP as phosphoryl donors, whereas ATP-NAD kinase is specific for ATP. Poly(P)/ATP-NAD kinase has been identified in the prokaryotes of the actinobacteria group, such as Mycobacterium tuberculosis and Micrococcus flavus, and is designated as Ppnk (M. tuberculosis) and Mfnk (M. flavus) (5). ATP-NAD kinase has been identified in the prokaryotes of the proteobacteria group such as Escherichia coli and Sphingomonas sp. A1 and in eukaryotes such as the yeast Saccharomyces cerevisiae and are designated as YfjB (E. coli); NadK (Sphingomonas sp. A1); and Utr1p, Yef1p, and Pos5p (S. cerevisiae) 2 (2, 6, 8 -10). Among the NAD kinases, the te...

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Section: Nadmentioning

confidence: 99%

Section: Nadmentioning

confidence: 99%

Molecular Conversion of NAD Kinase to NADH Kinase through Single Amino Acid Residue Substitution

Mori

Kawai

Shi

et al. 2005

Journal of Biological Chemistry

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“…The NAD kinase gene was later cloned from M. flavus. 22) Ppnk is the enzyme whose structure has been studied in detail by us [23][24][25][26] and by other groups, 27,28) as described below. Identification of the NAD kinase gene prompted us to search for NAD kinase homolog genes in the genome sequences of all organisms, including archaea, eubacteria, and eucaryotes, with the exception of the intracellular parasite Chlamydia trachomatis, which has no pyridine metabolism gene homolog.…”

Section: Identification and Molecular Properties Of Nad Kinasementioning

confidence: 99%

“…PCC6301, and yeast S. cerevisiae have three homolog genes; and the yeast Candida albicans, the protist (a cellular slime mold) Dictyostelium discoideum, and some eubacteria (Bacillus, Listeria, Streptomyces, and other cyanobacteria) contain two homolog genes. We cloned NAD kinase homolog genes from several microorganisms, 21,22,26,[30][31][32][33][34] and other researchers have cloned them from microorganisms, 3,29,[35][36][37] plants, [38][39][40][41] and humans, 42) and the properties of the gene products (recombinant NAD kinases) were determined (summarized in Table 1). All characterized NAD kinases show homomultimer (homodimer, homotetramer, homohexamer, or homooctamer) structures.…”

Section: Identification and Molecular Properties Of Nad Kinasementioning

confidence: 99%

Structure and Function of NAD Kinase and NADP Phosphatase: Key Enzymes That Regulate the Intracellular Balance of NAD(H) and NADP(H)

Kawai

Murata

2008

Bioscience, Biotechnology, and Biochemistry

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þ and NADPH) are undoubtedly significant and distinct. Hence, regulation of the intracellular balance of NAD(H) and NADP(H) is important. The key enzymes involved in the regulation are NAD kinase and NADP phosphatase. In 2000, we first succeeded in identifying the gene for NAD kinase, thereby facilitating worldwide studies of this enzyme from various organisms, including eubacteria, archaea, yeast, plants, and humans. Molecular biological study has revealed the physiological function of this enzyme, that is to say, the significance of NADP(H), in some model organisms. Structural research has elucidated the tertiary structure of the enzyme, the details of substratebinding sites, and the catalytic mechanism. Research on NAD kinase also led to the discovery of archaeal NADP phosphatase. In this review, we summarize the physiological functions, applications, and structure of NAD kinase, and the way we discovered archaeal NADP phosphatase.

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“…(20), and Bacillus subtilis (6). The NAD kinase genes from some bacteria, such as M. tuberculosis (10), M. flavus (11), and Escherichia coli (9), have been cloned and sequenced. Among them, NAD kinases from many organisms, except for a few bacteria, utilize ATP and some other nucleoside triphosphates, but not inorganic polyphosphate [poly(P)], as phosphate donors.…”

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confidence: 99%

First Archaeal Inorganic Polyphosphate/ATP-Dependent NAD Kinase, from Hyperthermophilic Archaeon Pyrococcus horikoshii : Cloning, Expression, and Characterization

Sakuraba

Kawakami

Ohshima

2005

Appl Environ Microbiol

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The gene (PH1074) encoding the NAD kinase of the hyperthermophilic archaeon Pyrococcus horikoshii was identified in the genome database, cloned, and functionally expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment at 90°C for 20 min and one successive HiTrap affinity chromatography step. The purified enzyme was easily precipitated by dialysis against phosphate buffer without NaCl and imidazole and was usually stored in buffer containing 0.5 M NaCl and 0.5 M imidazole to avoid precipitation. The molecular mass of the active enzyme was determined to be 145 kDa by a gel filtration method, and the enzyme was composed of a tetramer of 37-kDa subunits. The archaeal enzyme utilized several nucleoside triphosphates, such as GTP, CTP, UTP, and ITP, as well as ATP and inorganic polyphosphates [poly(P)] as phosphoryl donors for NAD phosphorylation. The enzyme utilized poly(P) 27 (the average length of the phosphoryl chain was 27) as the most active inorganic polyphosphate for NAD phosphorylation. Thus, this enzyme is categorized as an inorganic polyphosphate/ATP-dependent NAD kinase. The enzyme was the most thermostable NAD kinase found to date: its activity was not lost by incubation at 95°C for 10 min. The enzyme showed classical Michaelis-Menten-type kinetics for NAD and ATP, but not for poly(P) 27 . The K m values for NAD were determined to be 0.30 and 0.40 mM when poly(P) 27 and ATP, respectively, were used as the phosphoryl donors. The K m value for ATP was 0.29 mM, and the concentration of poly(P) 27 which gave half of the maximum enzyme activity was 0.59 mM. The enzyme required several metal cations, such as Mg 20), and Bacillus subtilis (6). The NAD kinase genes from some bacteria, such as M. tuberculosis (10), M. flavus (11), and Escherichia coli (9), have been cloned and sequenced. Among them, NAD kinases from many organisms, except for a few bacteria, utilize ATP and some other nucleoside triphosphates, but not inorganic polyphosphate [poly(P)], as phosphate donors. On the other hand, the enzymes from M. tuberculosis, M. flavus, and B. subtilis have been reported to utilize inorganic polyphosphate [poly(P)] as well as ATP for NAD phosphorylation (6, 10). Poly(P) is a polymer of inorganic orthophosphate residues linked by high-energy phosphoanhydride bonds and is proposed to be a primitive energy source for living organisms (14). Although poly(P)/ATP-NAD kinases are postulated to be prototypes of ATP-dependent NAD kinases, there is no information so far about NAD kinases from archaea, which form the third and evolutionally oldest domain of life (25). Poly(P) could be used as a phosphoryl donor for enzymatic phosphorylation instead of ATP and ADP. NAD kinases from mesophiles have been used for the industrial production of NADP from NAD (8), and poly(P) is commercially available as a much less expensive phosphoryl donor than ATP. Stable poly(P)-dependent NAD kinase is more useful for enzymatic NADP production. The hyperthermophilic archaea have a high potential as a...

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

Cited by 15 publications

References 14 publications

Molecular Conversion of NAD Kinase to NADH Kinase through Single Amino Acid Residue Substitution

Molecular Conversion of NAD Kinase to NADH Kinase through Single Amino Acid Residue Substitution

Structure and Function of NAD Kinase and NADP Phosphatase: Key Enzymes That Regulate the Intracellular Balance of NAD(H) and NADP(H)

First Archaeal Inorganic Polyphosphate/ATP-Dependent NAD Kinase, from Hyperthermophilic Archaeon Pyrococcus horikoshii : Cloning, Expression, and Characterization

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