Assimilation of Nicotinamide Mononucleotide Requires Periplasmic AphA Phosphatase inSalmonella enterica

Abstract

Salmonella enterica can obtain pyridine from exogenous nicotinamide mononucleotide (NMN) by three routes. In route 1, nicotinamide is removed from NMN in the periplasm and enters the cell as the free base. In route 2, described here, phosphate is removed from NMN in the periplasm by acid phosphatase (AphA), and the produced nicotinamide ribonucleoside (NmR) enters the cell via the PnuC transporter. Internal NmR is then converted back to NMN by the NmR kinase activity of NadR. Route 3 is seen only in pnuC* transporter mutants, which import NMN intact and can therefore grow on lower levels of NMN. Internal NMN produced by either route 2 or route 3 is deamidated to nicotinic acid mononucleotide and converted to NAD by the biosynthetic enzymes NadD and NadE.

Structure, function, evolution and application of bacterial Pnu-type vitamin transporters

“…NR is one of several related compounds (vitamers) that have been named vitamin B3 and is a precursor of the cofactor NAD (Figure 1), which is found in all living organisms. The specificity of PnuC for NR has been demonstrated for homologs from Escherichia coli (Sauer et al, 2004), Haemophilus influenzae (Kemmer et al, 2001), Haemophilus parainfluenzae (Cynamon et al, 1988), S. typhimurium (Grose et al, 2005b), and Neisseria mucosa (Jaehme et al, 2014). In contrast to other Pnu homologs, the genomic organization of the genes encoding PnuC is not well conserved.…”

“…However, subsequent research showed that both transporters are similar and also use a similar mode of transport. In both organisms, periplasmic machineries have been identified that breakdown NAD and NMN to NR, which is the transported substrate of PnuC (Cynamon et al, 1988;Kemmer et al, 2001;Herbert et al, 2003;Sauer et al, 2004;Grose et al, 2005b). Shortly after cloning of PnuC, it became evident that its activity strongly depends on the regulator NadR (Foster et al, 1987(Foster et al, , 1990Zhu et al, 1989.…”

“…The outer membrane lipoprotein e (P4) has an NMN 5′-nucleotidase activity, and the periplasmic NAD-nucleotidase NadN has an NAD pyrophosphatase and an NMN 5′-nucleotidase activity (Kemmer et al, 2001). Similarly, in Salmonella enterica, the periplasmic phosphatase AphA removes the phosphate group from NMN (Grose et al, 2005b). Thus, these proteins are essential to use external NAD and NMN sources.…”

“…For PnuC as well as for PnuX, experimental evidence exists for excretion of their substrates when the internal concentrations are high (Grose et al, 2005b;Hemberger et al, 2011) Pyridine excretion might be a physiological requirement under certain conditions as the bacterial DNA-ligase is inhibited by NMN (Zimmerman et al, 1967). PnuC as a facilitator could establish an exit route for an excess of pyridines.…”

“…The gene encoding PnuC from Salmonella typhimurium was cloned in 1986, and the protein was initially designated as the transporter of a previously characterized nicotinamide mononucleotide (NMN) uptake activity (Kinney et al, 1979;Liu et al, 1982;Spector et al, 1985;Tirgari et al, 1986;. Later studies showed that the substrate for PnuC is not NMN but its unphosphorylated precursor nicotinamide riboside (NR), which is one of the manifestations of vitamin B3, besides nicotinamide and nicotinic acid (Kemmer et al, 2001;Bieganowski and Brenner, 2004;Grose et al, 2005b). With the help of comparative genomics approaches, homologs of PnuC with varying substrate specificities were predicted in the last decade Vitreschak et al, 2002;Rodionov and Gelfand, 2005).…”