Abstract
NAD kinase phosphorylates NAD(+) to form NADP(+). Conversely, NADP phosphatase, which has not yet been identified, dephosphorylates NADP(+) to produce NAD(+). Among the NAD kinase homologs, the primary structure of MJ0917 of hyperthermophilic archaeal Methanococcus jannaschii is unique. MJ0917 possesses an NAD kinase homologous region in its C-terminal half and an inositol-1-phosphatase homologous region in its N-terminal half. In this study, MJ0917 was biochemically shown to possess both NAD kinase and phosphatase activities toward NADP(+), NADPH, and fructose 1,6-bisphosphate, but not toward inositol 1-phosphate. With regard to the phosphatase activity, kinetic values indicated that NADP(+) is the preferred substrate and that MJ0917 would function as a novel NADP phosphatase/NAD kinase showing conflicting dual activities, viz. synthesis and degradation of an essential NADP(+). Furthermore, in vitro analysis of MJ0917 showed that, although MJ0917 could supply NADP(+), it prevented excess accumulation of NADP(+); thus, it has the ability to maintain a high NAD(+)/NADP(+) ratio, whereas 5'-AMP would decrease this ratio. The evolutionary process during which MJ0917 arose is also discussed.
Highlights
A key enzyme regulating the balance of NADϩ/NADPϩ is NAD kinase (EC 2.7.1.23); it phosphorylates NADϩ to form NADPϩ
NADP phosphatase (NADPase) activity has been detected in rat liver mitochondria [4] and in bacteria [5], and it was found to correlate with the circadian rhythm of Euglena [6] and with seed dormancy in Avena sativa L. [7]
SDS-PAGE of the E. coli cell extracts showed that MJ0917 (Fig. 1B, lane 3) and MJ0917-C were expressed as soluble proteins, but that MJ0917-N was expressed only as an insoluble protein
Summary
Assays—ATP-dependent NAD kinase activity was assayed in a 1.0-ml reaction mixture (5.0 mM NADϩ, 5.0 mM ATP, 20 mM MgCl2, and 100 mM Tris-HCl (pH 8.5)) at 85 °C [12]. In all assays for NAD kinase and phosphatase, reactions were initiated by the addition of the enzyme to reaction mixtures preincubated at 85 °C for 3 min. After removing aggregated proteins by centrifugation at 20,000 ϫ g for 10 min, the supernatant was applied to a Toyopearl AF-Blue HC-650 M column (1.0 ϫ 3.2 cm; Tosoh Corp., Tokyo) equilibrated with TE buffer containing 10 mM MgCl2 at 4 °C and eluted with a linear gradient of NaCl (0 –3000 mM) in the same buffer (40 ml). MJ0917-C was partially purified by heating the cell extract containing MJ0917-C at 85 °C for 2.5 min and removing the aggregated proteins as described above. BLAST homology analysis [30] was conducted on the GenomeNet web site (available at blast.genome.jp/) against the Kyoto Encyclopedia of Genes and Genomes GENES plus DGENES
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