No abstract

NAD kinase is the sole NADP+ biosynthetic enzyme. Despite the great significance of NADP+, to date no mitochondrial NAD kinase has been identified in human, and the source of human mitochondrial NADP+ remains elusive. Here we present evidence demonstrating that a human protein of unknown function, C5orf33, is a human mitochondrial NAD kinase; this protein likely represents the missing source of human mitochondrial NADP+. The C5orf33 protein exhibits NAD kinase activity, utilizing ATP or inorganic polyphosphate, and is localized in the mitochondria of human HEK293A cells. C5orf33 mRNA is more abundant than human cytosolic NAD kinase mRNA in almost all tissues examined. We further show by database searches that some animals and protists carry C5orf33 homologues as their sole NADP+ biosynthetic enzyme, whereas plants and fungi possess no C5orf33 homologue. These observations provide insights into eukaryotic NADP+ biosynthesis, which has pivotal roles in cells and organelles.

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...