title: NADPH regulates human NAD kinase, a NADP⁺-biosynthetic enzyme.
abstract: NAD kinase (NADK, EC 2.7.1.23) is the sole NADP(+)-biosynthetic enzyme that catalyzes phosphorylation of NAD(+) to yield NADP(+) using ATP as a phosphoryl donor, and thus, plays a vital role in the cell and represents a potentially powerful antimicrobial drug target. Although methods for expression and purification of human NADK have been previously established (Lerner et al. Biochem Biophys Res Commun 288:69-74, 2001), the purification procedure could be significantly improved. In this study, we improved the method for expression and purification of human NADK in Escherichia coli and obtained a purified homogeneous enzyme only through heat treatment and single column chromatography. Using the purified human NADK, we revealed a sigmoidal kinetic behavior toward ATP and the inhibitory effects of NADPH and NADH, but not of NADP(+), on the catalytic activity of the enzyme. These inhibitory effects provide insight into the regulation of intracellular NADPH synthesis. Furthermore, these attributes may provide a clue to design a novel drug against Mycobacterium tuberculosis in which this bacterial NADK is potently inhibited by NADP(+).
authors.affiliation: Laboratory of Basic and Applied Molecular Biotechnology, Division of Food and Biological Science, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
authors.forename: Kazuto
authors.initials: K
authors.lastname: Ohashi
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Kawai
authors.forename: Mari
authors.initials: M
authors.lastname: Koshimizu
authors.forename: Kousaku
authors.initials: K
authors.lastname: Murata
mesh_terms: D000890:Anti-Infective Agents / Q000737:chemistry; D003001:Cloning, Molecular; D015195:Drug Design; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000737:chemistry; D004926:Escherichia coli; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D009243:NAD / Q000737:chemistry; D009249:NADP / Q000096:biosynthesis / Q000737:chemistry*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D000890:Anti-Infective Agents; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; D017853:Phosphotransferases (Alcohol Group Acceptor); C021844:NAD kinase