NAD kinase controls animal NADP biosynthesis and is modulated via evolutionarily divergent calmodulin-dependent mechanisms

Abstract

Nicotinamide adenine dinucleotide phosphate (NADP) is a critical cofactor during metabolism, calcium signaling, and oxidative defense, yet how animals regulate their NADP pools in vivo and how NADP-synthesizing enzymes are regulated have long remained unknown. Here we show that expression of Nadk, an NAD + kinase-encoding gene, governs NADP biosynthesis in vivo and is essential for development in Xenopus frog embryos. Unexpectedly, we found that embryonic Nadk expression is dynamic, showing cell type-specific up-regulation during both frog and sea urchin embryogenesis. We analyzed the NAD kinases (NADKs) of a variety of deuterostome animals, finding two conserved internal domains forming a catalytic core but a highly divergent N terminus. One type of N terminus (found in basal species such as the sea urchin) mediates direct catalytic activation of NADK by Ca 2+ /calmodulin (CaM), whereas the other (typical for vertebrates) is phosphorylated by a CaM kinase-dependent mechanism. This work indicates that animal NADKs govern NADP biosynthesis in vivo and are regulated by evolutionarily divergent and conserved CaM-dependent mechanisms. + and reduced form NADPH) is one of the most fundamental metabolic molecules inside cells. NADPH serves essential functions in reductive biosyntheses, detoxification processes (mediated by cytochrome P450), and the regeneration of oxidative defense systems. Moreover, it acts as an electron donor for NADPH oxidases, which generate reactive oxygen species for immune cell respiratory bursts and for redox signaling (1). NADP + can also be converted to NAADP, a potent intracellular calcium messenger (2). However, despite its importance for cellular function, surprisingly little is known about how animals regulate their cellular NADP pools.Earlier work has shown that animals use vitamin B3 to produce NAD, and in vitro studies have established that NAD can be phosphorylated to yield NADP by NAD kinase (NADK) enzymes using ATP as a phosphoryl donor (3-5) (Fig. 1A). However, there have been no in vivo studies examining the function of animal NADK genes, and thus whether NADK is required for sustaining NADP levels in vivo has remained unknown.In addition, the mechanisms governing the regulation of the NADK enzyme are not well-understood. Previous studies, however, have demonstrated a species-dependent differential response of NADK activity following exposure to calcium/calmodulin (Ca 2+ /CaM). For instance, NADK activity in plant extracts is increased by the addition of Ca 2+ /CaM (6). However, none of the three recombinantly expressed NADK isoforms from Arabidopsis thaliana were responsive to Ca 2+ /CaM (7,8). In animals, a Ca 2+ / CaM-dependent rise of NADP following sea urchin egg fertilization was observed more than 30 y ago, although the molecular mechanisms underlying this phenomenon have not been elucidated.We previously identified and characterized the human NADK encoded by the gene Nadk (1, 9), finding its enzymatic activity insensitive to Ca 2+ /CaM. Thus, the interaction between the N...

Dexamethasone improves redox state in ataxia telangiectasia cells by promoting an NRF2‐mediated antioxidant response

“…NADK activity was measured essentially as described previously . NADK catalyzes the conversion of NAD + to NADP + using ATP as phosphate group donor: …”

“…NADK activity was measured essentially as described previously [73]. NADK catalyzes the conversion of NAD + to NADP + using ATP as phosphate group donor:…”

Integration of nicotinic acid adenine dinucleotide phosphate (NAADP)‐dependent calcium signalling

“…Currently, two routes have been suggested as synthetic pathways in vitro. Starting from NAD, its phosphorylated derivative NADP is synthesized by NAD kinase (Pollak et al 2007;Love et al 2015; Fig. 1).…”

Clinical heterogeneity of mitochondrial NAD kinase deficiency caused by a NADK2 start loss variant

“…Other additional NADPH dependent enzymes may include the Aldose Reductase dependent reduction of glucose into sorbitol, the malic enzyme dependent reductive carboxylation of pyruvate, the isocitrate dehydrogenase dependent reductive carboxylation in the citric acid cycle, and the glutathione reductase dependent mitochondrial metabolism of reactive oxygen species (Love et al, 2015; Tedeschi et al, 2016). Symptoms of other mitochondrial disorders can range from clinically mild, like lactic acidosis, peripheral neuropathy and optic neuropathy to more severe symptoms such as renal tubular acidosis, hypotonia, developmental delay, progressive encephalopathy, and failure to thrive (Zhang, 2015).…”

“…It is also phosphorylated by NADK2 to produce NADPH (Love et al, 2015). The instability of NADPH, as well as its limited permeability across cellular membranes, suggests that it is unlikely to be beneficial as a nutrient supplement (Wu, Wu, & Knight, 1986).…”