NADP+/NADPH

The NADP⁺/NADPH cofactor pair is essential for reductive biosyntheses and the photosynthesis in plant chloroplasts. The reduced form, NADPH plays also an important role in detoxification reactions and cellular defense mechanisms against oxidative stress. That is, reductases maintaining the reduced pool of both, glutathione and thioredoxin, utilize NADPH as cofactor.

NADP is produced from NAD catalyzed by NAD kinase (NADK). This reaction represents the only known de novo way to generate NADP both in prokaryotic and eukaryotic cells (Fig. 1). NADK has been studied for decades in various organisms but it was only recently that conclusive structural information has become available. Based on the identified amino acid sequences of the enzyme from Micrococcus flavus and Mycobacterium tuberculosis, cDNAs encoding NADKs were cloned from bacteria, yeast, plant and human (for reviews see Magni et al., 2006 and Pollak et al., 2007a). In yeast and plants, three NADK isoforms were identified showing distinct substrate specificities and subcellular localization. Strikingly, so far only a single isoform has been found in mammals and only little is known about the regulation of its activity to maintain the cellular NADP pool.

Our studies focus on the functional significance of the known human NADK. The enzyme was found to be localized to the cytoplasm and preferentially to accept NAD⁺ as substrate (Pollak et al., 2007b). We have generated cell lines stably overexpressing human NADK or small hairpin RNA targeting the NADK mRNA. Using these cell systems we have shown that modulation of NADK expression is primarily reflected in the alteration of the NADPH, and not NADP⁺, levels. However, even though the NADPH concentration is increased 4-5 fold, the strong overexpression of NADK provides only moderate protection upon challenging the cells with oxidizing agents. Modulation of NADK expression and according the cellular NADPH content influences the expression of two proteins involved in oxidative stress response, namely PRDX5 and Nrf2. Our results point to a direct relationship between the total cellular NADPH content and activation of anti-oxidative mechanisms (Fig. 2). Currently we are interested in the mechanism(s) linking NADK and accordingly, NADPH levels to PRDX5 and Nrf2 expression.

Calcium/calmodulin stimulates the enzymes from plants, sea urchin eggs and human neutrophils. Due to the importance of cellular calcium signalling this activation could represent a key regulatory step. So far, the molecular mechanism of the calcium/calmodulin-dependent activation of NADKs has remained unknown. None of the purified recombinant NADKs from plant Arabidopsis thaliana nor the human enzyme were directly affected by calcium/calmodulin. These observations suggest that the reported activation of NADK is indirect and could require additional factors.

Previously we have identified two NADK isoforms of the sea urchin Strongylocentrotus purpuratus and demonstrated these NADKs to be directly activated by calcium/calmodulin (Pollak et al., 2007c). Furthermore, we have shown that isoform 1 is phosphorylated by CaMKII suggesting the existence of an additional calcium/calmodulin-mediated pathway to regulate NADK activity. Currently we are interested in the possibility of such a mechanism for human NADK.

References:

Magni, G., Orsomando, G. and Raffaelli, N. (2006) Medicinal Chemistry 6

Pollak, N., Dölle, C. and Ziegler, M. (2007a) Biochemical Journal 402

Pollak, N., Niere, M. and Ziegler, M. (2007b) Journal of Biological Chemistry 282

Pollak, N., Niere, M., Patel, S. and Ziegler, M. (2007c)