Identification and characterization of a human mitochondrial NAD kinase

Abstract

NAD kinase is the sole NADP+ biosynthetic enzyme. Despite the great significance of NADP+, to date no mitochondrial NAD kinase has been identified in human, and the source of human mitochondrial NADP+ remains elusive. Here we present evidence demonstrating that a human protein of unknown function, C5orf33, is a human mitochondrial NAD kinase; this protein likely represents the missing source of human mitochondrial NADP+. The C5orf33 protein exhibits NAD kinase activity, utilizing ATP or inorganic polyphosphate, and is localized in the mitochondria of human HEK293A cells. C5orf33 mRNA is more abundant than human cytosolic NAD kinase mRNA in almost all tissues examined. We further show by database searches that some animals and protists carry C5orf33 homologues as their sole NADP+ biosynthetic enzyme, whereas plants and fungi possess no C5orf33 homologue. These observations provide insights into eukaryotic NADP+ biosynthesis, which has pivotal roles in cells and organelles.

Suppression of Cytosolic NADPH Pool by Thionicotinamide Increases Oxidative Stress and Synergizes with Chemotherapy

“…Though we focused on the effects of inhibiting cytosolic NADK in this study, it is important to consider the newly discovered and characterized mitochondrial NADK (mNADK) (Ohashi et al, 2012;Zhang, 2015). In a previous study, Zhang (2015) found mNADK had lower activity compared with NADK and fact had lower expression in liver tumor samples, in contrast with the overexpression of NADK in a variety of cancer types (unpublished data).…”

Evidence that the antiproliferative effects of auranofin in Saccharomyces cerevisiae arise from inhibition of mitochondrial respiration

“…Although Pos5 shows a low primary structure homology with the human enzyme, the tertiary structure and the key aminoacids of the NADH binding site appear conserved (Ando et al, 2011). Recently, the human mitochondrial NADH kinase (NADK2) has been identified (Ohashi et al, 2012) and mitochondrial NADP(H) deficiency due to a mutation in NADK2 was also proposed as cause of mitochondrial disorders (Houten et al, 2014). Further studies will be necessary to investigate if the increased resistance of other identified deleted strains is a consequence of Pos5 inactivation or is due to a direct activity of AF.…”

Fe-S Cluster Biogenesis in Isolated Mammalian Mitochondria

“…The NAD(H) kinases are the sole enzymes able to convert NAD(H) to NADP(H) using ATP as a phosphate donor. Such a kinase has been identified recently in mammalian mitochondria (51,52), although a possible functional connection between the kinase and Fe-S cluster biogenesis remains to be determined. By contrast, the corresponding NADH kinase in the yeast mitochondrial matrix, called Pos5, has been characterized, and it seems that NADPH, generated by Pos5, plays an important role in Fe-S cluster biogenesis in yeast mitochondria (17,53).…”

“…The reductase is likely to be adrenodoxin reductase, which reduces ferredoxins FDX1 and FDX2 (38,39). The reducing equivalents for this electron transport chain may derive from NADPH (50), and, therefore an NAD(H) kinase may also be required inside mitochondria (17,51). The substrate that receives electrons is unknown but could be the S 0 and/or iron involved in Fe-S cluster intermediate formation (4).…”