We ran PSI-BLAST (Altschul et al., 1997) on the predicted S. cerevisiae Nrk1 polypeptide and discovered the apparent orthologous human protein Nrk1 (locus NP_060351) encoded at 9q21.31, encoding a polypeptide of 199 amino acids annotated as an uncharacterized protein of the uridine kinase family. In addition, we found a second human gene product Nrk2 (locus NP_733778) that is 57% identical to human Nrk1. Nrk2 is a 230 amino acid splice form of what was described as a 186 amino acid muscle integrin β1 binding protein (ITGB1BP3) encoded at 19p13.3 (Li et al. 1999, Li et al. 2003). Amino acid conservation between S. cerevisiae, S. pombe, and human Nrk homologs and similarity with fragments of S. cerevisiae Urk1 and E. coli panK is shown in Figure 4B. As shown in Figure 4C, complementation of the failure of qns1 nrk1 to grow on nicotinamide riboside-supplemented media was provided by human NRK1 and human NRK2 cDNAs expressed from the yeast GAL1 promoter.
Purification of yeast Nrk1 and human Nrk1 and Nrk2 revealed high specificity for phosphorylation of nicotinamide riboside and tiazofurin (Table 1). In the cases of yeast and human Nrk1, the enzymes actually prefer tiazofurin to the natural substrate nicotinamide riboside by a factor of two and both enzymes retain less than 7% of their maximal specific activity on uridine and cytidine. In the case of human Nrk2, the 186 amino acid integrin β1 binding protein form is devoid of enzymatic activity (data not shown). On the other hand, the 230 amino acid form is essentially equally active on nicotinamide riboside, tiazofurin, and uridine with less than 10% of corresponding activity on cytidine. Thus, though Nrk2 may contribute additionally to formation of uridylate in the tissues in which it is expressed, these data demonstrate that fungi and mammals possess specific Nrks that function to synthesize NAD+ through NMN in addition to the well-known pathways through NaMN. Identification of Nrk enzymatic activities thus accounts for the dual specificity of fungal and mammalian NaMN/NMN adenylyltransferases.