NADPH regulates human NAD kinase, a NADP⁺-biosynthetic enzyme.
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NAD kinase (NADK, EC 18.104.22.168) 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(+).
- Molecular and cellular biochemistry
Molecular and cellular biochemistry 355(1-2), 57-64, 2011-09
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