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NADPH regulates human NAD kinase, a NADP+-biosynthetic enzyme

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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+.

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Abbreviations

NADK:

NAD kinase

LB:

Luria–Bertani

SDS-PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

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Acknowledgments

This study was supported in part by a Grant-in-Aid for SK from the Ministry of Education, Culture, Sports, Science and Technology of Japan (21780069).

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Authors and Affiliations

  1. Laboratory of Basic and Applied Molecular Biotechnology, Division of Food and Biological Science, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, 611-0011, Japan

    Kazuto Ohashi, Shigeyuki Kawai, Mari Koshimizu & Kousaku Murata

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  1. Kazuto Ohashi
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  2. Shigeyuki Kawai
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  3. Mari Koshimizu
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  4. Kousaku Murata
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Correspondence to Kousaku Murata.

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Ohashi, K., Kawai, S., Koshimizu, M. et al. NADPH regulates human NAD kinase, a NADP+-biosynthetic enzyme. Mol Cell Biochem 355, 57–64 (2011). https://doi.org/10.1007/s11010-011-0838-x

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  • DOI: https://doi.org/10.1007/s11010-011-0838-x

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