Ribosylnicotinamide Kinase Domain of NadR Protein: Identification and Implications in NAD Biosynthesis

ABSTRACTNAD is an indispensable redox cofactor in all organisms. Most of the genes required for NAD biosynthesis in various species are known. Ribosylnicotinamide kinase (RNK) was among the few unknown (missing) genes involved with NAD salvage and recycling pathways. Using a comparative genome analysis involving reconstruction of NAD metabolism from genomic data, we predicted and experimentally verified that bacterial RNK is encoded within the 3′ region of thenadRgene. Based on these results and previous data, the full-size multifunctional NadR protein (as inEscherichia coli) is composed of (i) an N-terminal DNA-binding domain involved in the transcriptional regulation of NAD biosynthesis, (ii) a central nicotinamide mononucleotide adenylyltransferase (NMNAT) domain, and (iii) a C-terminal RNK domain. The RNK and NMNAT enzymatic activities of recombinant NadR proteins fromSalmonella entericaserovar Typhimurium andHaemophilus influenzaewere quantitatively characterized. We propose a model for the complete salvage pathway from exogenousN-ribosylnicotinamide to NAD which involves the concerted action of the PnuC transporter and NRK, followed by the NMNAT activity of the NadR protein. Both thepnuCandnadRgenes were proven to be essential for the growth and survival ofH. influenzae, thus implicating them as potential narrow-spectrum drug targets.