Molecular characterization of Escherichia coli NAD kinase

Abstract

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...

Cloning and Characterization of Two NAD Kinases from Arabidopsis. Identification of a Calmodulin Binding Isoform

“…Although considerable variability between plant species has been reported for the V max of plant CaMdependent NADKs, the specific activity of native Arabidopsis CaM-dependent NADK is within the range described for partially pure NADKs from plants such as pea (Harmon et al, 1984) and tomato (Delumeau et al, 1998). Similarly, the pH optimum (pH 8.0) for Arabidopsis NADKs (Table II) is comparable to that for human (Lerner et al, 2001), yeast (Kawai et al, 2001b), and E. coli (Kawai et al, 2001a) NADKs as well as other plant NADKs (Muto, 1983;Gallais et al, 2001). The K m values for native CaM-dependent NADK, for both Mg 21 -ATP (200 mM) and NAD (170 mM), are comparable to reports on CaMdependent NADKs from pea (Muto, 1983), Avena sativa , and tomato, with the exception that the tomato enzyme appears to have a much higher affinity for Mg 21 -ATP (Delumeau et al, 1998).…”

“…With the exception of the conserved C-terminal region, no other identifiable structural motifs were predicted in either NADK1 or NADK2. A more detailed sequence alignment of the C-terminal regions, believed to include the catalytic domain (Kawai et al, 2001a), for various NADKs is presented in Figure 1B. The NADK motifs, XXX-XXGGDG-XL and GDXXX-TPTGSTAY (where X represents a hydrophobic residue) are also largely conserved in both NADK1 and NADK2 (Kawai et al, 2001a).…”

“…A more detailed sequence alignment of the C-terminal regions, believed to include the catalytic domain (Kawai et al, 2001a), for various NADKs is presented in Figure 1B. The NADK motifs, XXX-XXGGDG-XL and GDXXX-TPTGSTAY (where X represents a hydrophobic residue) are also largely conserved in both NADK1 and NADK2 (Kawai et al, 2001a). It is noteworthy that the genomes of yeast (Kawai et al, 2001b;Outten and Culotta, 2003) and Arabidopsis (this work) predict multiple NADKs, whereas only a single NADK has been described in humans (Lerner et al, 2001).…”

“…NADKs have been found in all organisms examined to date, suggesting a fundamental role for the enzyme (McGuinness and Butler, 1985;Zielinski, 1998;Kawai et al, 2001aKawai et al, , 2001bLerner et al, 2001). While the importance of the pyridine nucleotides NAD(H) and NADP(H) in energy metabolism and reductive biosynthesis is well established, the role of NADK itself remains unclear.…”

“…While the importance of the pyridine nucleotides NAD(H) and NADP(H) in energy metabolism and reductive biosynthesis is well established, the role of NADK itself remains unclear. Furthermore, while the enzyme has been studied for decades in various organisms, it was only recently that cDNAs encoding NADKs were cloned from bacteria (Kawai et al, 2000(Kawai et al, , 2001a, yeast (Kawai et al, 2001b), and human (Lerner et al, 2001) sources. Until this study, a cDNA encoding a plant NADK had not been isolated, and the lack of molecular tools has hindered research progress on the roles of NADKs in plants.…”