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4.B.1 The Nicotinamide Ribonucleoside (NR) Uptake Permease (PnuC) Family

PnuC of Salmonella typhimurium and Haemophilus influenzae are integral membrane proteins, 239 and 226 amino acyl residues (aas) in length, respectively, with 7 putative transmembrane α-helical segments (TMSs). They are believed to function cooperatively with NadR homologues, multifunctional proteins that together with PnuC, participate in NR phosphorylation, transport and transcriptional regulation (Foster et al., 1990; Merdanovic et al., 2005; Penfound et al., 1999). NadR, a cytoplasmic protein that is partly membrane associated, contains one well conserved and one poorly conserved mononucleotide-binding consensus sequences (G-X4 GKS). It drives transport and may render transport responsive to internal pyridine nucleotide levels. While its N-terminal half functions as a repressor, its C-terminal half functions as an NR kinase in a putative group translocation process (Roseman, 1975). PnuC has been shown to resemble SWEET porters in overall fold (Jaehme et al. 2016), supporting the conclusion that these two families are members of the TOG superfamily (Yee et al. 2013). Close PnuC homologues are found in a wide range of Gram-negative and Gram-positive bacteria but not in archaea or eukaryotes.

The H. influenzae homologue has been shown to transport NR from the periplasm into the cytoplasm. Phosphorylation of NR by NadR is required for NR uptake. The ribonucleoside kinase (RNK) domain has both Walker A and Walker B motifs, responsible for ATP binding and phosphoryl transfer. In addition, a proposed LID domain was identified in RNK. LID domains have been found in other kinases, and these domains are regions which are able to move after substrate binding. They are responsible for coordination of three distinct conformations, an open state in the absence of substrate, a partially closed state after substrate binding, and a fully closed state when both substrates are present.

The structure of NadR has been determined (Singh et al., 2002). Mutations in the nadR gene which interfere with NR uptake occur in the C-terminal part of NadR (Foster and Penfound, 1993). A helix-turn-helix DNA binding domain present in NadR of S. enterica serovar Typhimurium (Foster and Penfound, 1993) could not be found in the NadR homologue of H. influenzae. Therefore, it was proposed that in H. influenzae NadR has no regulatory function at the transcriptional level (Kurnasov et al., 2002). The structures of the human NR kinase 1 (2QL6_P) with nucleotide and nucleoside substrates bound have been solved (Tempel et al., 2007). It is structurally similar to Rossmann fold metabolite kinases. The human enzyme is distantly related to the bacterial enzymes, nicotinamide riboside kinases, uridine kinases, uracil phosphoribosyl transferases, uridine monophosphate kinases and uridine/cytidine kinases.

In H. influenzae, NR enters the NAD+ resynthesis pathway after phosphorylation to NMN, and subsequently, NAD+ is synthesized from NMN and ATP via an NMN adenylyl transferase activity (Cynamon et al., 1988; Kurnasov et al., 2002). Summarizing these features, NadR represents an amazing multifunctional regulator/enzyme complex able to integrate several features, such as enzymatic catalysis, transport, and transcriptional regulatory activities.

The components of the H. influenzae pathway necessary for NAD+, NMN, and NR uptake were determined. Merdanovic et al. (2005) characterized two enzymes, an outer membrane nucleotide phosphatase, and an NAD+ nucleotidase (NadN) located in the periplasm (Kemmer et al., 2001; Reidl et al., 2000; Schmidt-Brauns et al., 2001). They showed that NAD+ and NMN cross the outer membrane mainly via the OmpP2 porin (Andersen et al., 2003).

Only NR can be utilized by the PnuC transport system located in the inner membrane (Herbert et al., 2003; Sauer et al., 2004). The pnuC gene product is the protein that is responsible for the main flow of the NR substrate into the cytoplasm. Also, H. influenzae pnuC knockout mutants are not able to grow in rats; these mutants do not show invasive growth in infected infant rats (Herbert et al., 2003). The study of Merdanovic et al. (2005) suggests that the RNK activity of NadR determines NR transport and is negatively regulated by cytoplasmic NAD+ feedback inhibition. Therefore, NR uptake is under NadR feedback control.

ATP, not the pmf, appears to be required for NR uptake. Thus, the driving force for NR uptake via PnuC is NR phosphorylation by NadR. A concerted group translocation mechanism can be considered whereby NadR facilitates the dissociation of NR from PnuC by phosphorylating it to NMN, thus preventing retrograde diffusion (efflux) of NR. The fact that NadR can be found as a soluble cytosolic protein as well as a membrane-associated protein may indicate that NadR and PnuC are in close proximity, which should be necessary to promote NR influx and to prevent efflux. If this is true, then substrate flow across the membrane is coupled to the rate of NR phosphorylation.

The proposed transport reaction catalyzed by PnuC and NadR is:

NR (out) + ATP (in) → NMN (in) + ADP (in).

References associated with 4.B.1 family:

Andersen, C., E. Maier, G. Kemmer, J. Blass, A.K. Hilpert, R. Benz, and J. Reidl. (2003). Porin OmpP2 of Haemophilus influenzae shows substrate specificity towards nicotinamide-derived nucleotide substrates. J. Biol. Chem. 278: 24269-24276. 12695515
Cynamon, M.H., T.B. Sorg, and A. Patapow. (1988). Utilization and metabolism of NAD by Haemophilus parainfluenzae. J. Gen. Microbiol. 134: 2789-2799. 3254936
Foster, J.W. and T. Penfound. (1993). The bifunctional NadR regulator of Salmonella typhimurium: location of regions involved with DNA binding, nucleotide transport and intramolecular communication. FEMS Microbiol. Lett. 112: 179-184. 8405960
Foster, J.W., Y.K. Park, T. Penfound, T. Fenger, and M.P. Spector. (1990). Regulation of NAD metabolism in Salmonella typhimurium: Molecular sequence analysis of the bifunctional nadR regulator and the nadA-pnuC operon. J. Bacteriol. 172: 4187-4196. 2198247
Herbert, M.A., E. Sauer, G. Smethurst, A. Kraiß, A.-K. Hilpert, and J. Reidl. (2003). Identification and characterization of nicotinamide-ribosyl uptake mutants in Haemophilus influenzae. Infect. Immun. 71: 5398-5401. 12933892
Jaehme, M., A. Guskov, and D.J. Slotboom. (2016). Pnu Transporters: Ain''t They SWEET? Trends. Biochem. Sci. 41: 117-118. 26692123
Jaehme, M., R. Singh, A.A. Garaeva, R.H. Duurkens, and D.J. Slotboom. (2018). PnuT uses a facilitated diffusion mechanism for thiamine uptake. J Gen Physiol 150: 41-50. 29203477
Kemmer, G., T.J. Reilly, J. Schmidt-Brauns, G.W. Zlotnik, B.A. Green, M.J. Fiske, M. Herbert, A. Kraiss, S. Schlör, A. Smith, and J. Reidl. (2001). NadN and e (P4) are essential for utilization of NAD and nicotinamide mononucleotide but not nicotinamide riboside in Haemophilus influenzae. J. Bacteriol. 183: 3974-3981. 11395461
Kurnasov, O.V., B.M. Polanuyer, S. Ananta, R. Sloutsky, A. Tam, S.Y. Gerdes, and A.L. Osterman. (2002). Ribosylnicotinamide kinase domain of NadR protein: identification and implications in NAD biosynthesis. J. Bacteriol. 184: 6906-6917. 12446641
Merdanovic, M., E. Sauer, and J. Reidl. (2005). Coupling of NAD+ biosynthesis and nicotinamide ribosyl transport: characterization of NadR ribonucleotide kinase mutants of Haemophilus influenzae. J. Bacteriol. 187: 4410-4420. 15968050
Penfound, T. and J.W. Foster. (1999). NAD-dependent DNA-binding activity of the bifunctional NadR regulator of Salmonella typhimurium. J. Bacteriol. 181: 648-655. 9882682
Penfound, T., and J. W. Foster. (1996). Biosynthesis and recycling of NAD, p. 721-730. In F.C. Neidhardt, R. Curtis III, J.L. Ingraham, E.C.C. Lin, K.B. Low, B. Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter, and H.E. Umbarger (ed.), Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd ed., vol. 1. American Society for Microbiology, Washington, D.C.
Reidl, J., S. Schlör, A. Kraiss, J. Schmidt-Brauns, G. Kemmer, and E. Soleva. (2000). NADP and NAD utilization in Haemophilus influenzae. Mol. Microbiol. 35: 1573-1581. 10760156
Rodionov, D.A. and M.S. Gelfand. (2005). Identification of a bacterial regulatory system for ribonucleotide reductases by phylogenetic profiling. Trends Genet. 21: 385-389. 15949864
Rodionov, D.A., A.G. Vitreschak, A.A. Mironov, and M.S. Gelfand. (2002). Comparative genomics of thiamin biosynthesis in procaryotes. New genes and regulatory mechanisms. J. Biol. Chem. 277: 48949-48959. 12376536
Rodionov, D.A., P. Hebbeln, A. Eudes, J. ter Beek, I.A. Rodionova, G.B. Erkens, D.J. Slotboom, M.S. Gelfand, A.L. Osterman, A.D. Hanson, and T. Eitinger. (2009). A novel class of modular transporters for vitamins in prokaryotes. J. Bacteriol. 191: 42-51. 18931129
Sauer, E., M. Merdanovic, A. Price Mortimer, G. Bringmann, and J. Reidl. (2004). Characterizing PnuC and the utilization of the nicotinamide riboside analog 3-aminopyridine in Haemophilus influenzae. Antimicrob. Agents Chemother. 48: 4532-4541. 15561822
Schmidt-Brauns, J., M. Herbert, G. Kemmer, A. Kraiss, S. Schlör, and J. Reidl. (2001). Is a NAD pyrophosphatase activity necessary for Haemophilus influenzae type b multiplication in the blood stream? Int. J. Med. Microbiol. 291: 219-225. 11554562
Singh, S.K., O.V. Kurnasov, B. Chen, H. Robinson, N.V. Grishin, A.L. Osterman, and H. Zhang. (2002). Crystal structure of Haemophilus influenzae NadR protein: a bifunctional enzyme endowed with NMN adenylyltransferase and ribosylnicotinamide kinase activities. J. Biol. Chem. 277: 33291-33299. 12068016
Tempel, W., W.M. Rabeh, K.L. Bogan, P. Belenky, M. Wojcik, H.F. Seidle, L. Nedyalkova1, T. Yang, A.A. Sauve, H.W. Park, C. Brenner. (2007). Nicotinamide Riboside Kinase Structures Reveal New Pathways to NAD+. PLoS Biol. 5(10):e263. 17914902
Vitreschak, A.G., D.A. Rodionov, A.A. Mironov, and M.S. Gelfand. (2002). Regulation of riboflavin biosynthesis and transport genes in bacteria by transcriptional and translational attenuation. Nucleic Acids Res 30: 3141-3151. 12136096
Vogl, C., S. Grill, O. Schilling, J. Stülke, M. Mack, and J. Stolz. (2007). Characterization of riboflavin (vitamin B2) transport proteins from Bacillus subtilis and Corynebacterium glutamicum. J. Bacteriol. 189: 7367-7375. 17693491
Yee, D.C., M.A. Shlykov, A. Västermark, V.S. Reddy, S. Arora, E.I. Sun, and M.H. Saier, Jr. (2013). The transporter-opsin-G protein-coupled receptor (TOG) superfamily. FEBS J. 280: 5780-5800. 23981446