Coupling of NAD+ biosynthesis and nicotinamide ribosyl transport: characterization of NadR ribonucleotide kinase mutants of Haemophilus influenzae.
Previously, we characterized a pathway necessary for the processing of NAD+ and for uptake of nicotinamide riboside (NR) in Haemophilus influenzae. Here we report on the role of NadR, which is essential for NAD+ utilization in this organism. Different NadR variants with a deleted ribonucleotide kinase domain or with a single amino acid change were characterized in vitro and in vivo with respect to cell viability, ribonucleotide kinase activity, and NR transport. The ribonucleotide kinase mutants were viable only in a nadV+ (nicotinamide phosphoribosyltransferase) background, indicating that the ribonucleotide kinase domain is essential for cell viability in H. influenzae. Mutations located in the Walker A and B motifs and the LID region resulted in deficiencies in both NR phosphorylation and NR uptake. The ribonucleotide kinase function of NadR was found to be feedback controlled by NAD+ under in vitro conditions and by NAD+ utilization in vivo. Taken together, our data demonstrate that the NR phosphorylation step is essential for both NR uptake across the inner membrane and NAD+ synthesis and is also involved in controlling the NAD+ biosynthesis rate.
Merdanovic
Melisa
M
Institut für Hygiene und Mikrobiologie, Universität Würzburg, Josef Schneider Str. 2, E1, 97080 Würzburg, Germany.
Sauer
Elizabeta
E
Reidl
Joachim
J
D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000378:metabolism*; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism; D006193:Haemophilus influenzae / Q000201:enzymology* / Q000235:genetics; D009154:Mutation; D009243:NAD / Q000096:biosynthesis*; D009536:Niacinamide / Q000031:analogs & derivatives* / Q000378:metabolism; D054409:Nicotinamide Phosphoribosyltransferase; D010430:Pentosyltransferases / Q000502:physiology*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000235:genetics; D021381:Protein Transport; D011726:Pyridinium Compounds; D012097:Repressor Proteins / Q000378:metabolism*; D012265:Ribonucleotides
D001426:Bacterial Proteins; C117105:NadR protein, bacteria; D011726:Pyridinium Compounds; D012097:Repressor Proteins; D012265:Ribonucleotides; C018613:nicotinamide-beta-riboside; D009243:NAD; D009536:Niacinamide; D010430:Pentosyltransferases; D054409:Nicotinamide Phosphoribosyltransferase; D010770:Phosphotransferases