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

Abstract

NAD 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 the nadR gene. Based on these results and previous data, the full-size multifunctional NadR protein (as in Escherichia 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 from Salmonella enterica serovar Typhimurium and Haemophilus influenzae were quantitatively characterized. We propose a model for the complete salvage pathway from exogenous N-ribosylnicotinamide to NAD which involves the concerted action of the PnuC transporter and NRK, followed by the NMNAT activity of the NadR protein. Both the pnuC and nadR genes were proven to be essential for the growth and survival of H. influenzae, thus implicating them as potential narrow-spectrum drug targets.

YCL047C/POF1 Is a Novel Nicotinamide Mononucleotide Adenylyltransferase (NMNAT) in Saccharomyces cerevisiae

“…Interestingly, Pof1 displayed an alkaline optimum pH (pH 10). Some NMNATs have been reported to be more active under alkaline conditions (29,30). It is possible that alkaline pH might affect Pof1 structure to mimic its in vivo state, thereby influencing the catalytic activity.…”

Microbial NAD Metabolism: Lessons from Comparative Genomics

“…The nadR gene, which is required for growth of H. influenzae on nicotin- (58). The nicotinamide riboside kinase domain of nadR is found in additional bacteria and is diagnostic for bacterial nicotinamide riboside utilization (33).…”

Structural Characterization of a Human Cytosolic NMN/NaMN Adenylyltransferase and Implication in Human NAD Biosynthesis

“…NMNAT-and NaMNAT-coupled Assay-The hsPNAT-3 activity with both substrates NMN and NaMN was analyzed using a continuous assay coupling NAD formation to alcohol dehydrogenase-catalyzed conversion of NAD to NADH as previously described (39,40). Briefly, the assay was performed in UV-transparent plastic cuvettes in the six-cuvette autosampler of a DU-640 spectrophotometer (Beckman, Fullerton, CA) at 37°C.…”