Coupling of NAD+Biosynthesis and Nicotinamide Ribosyl Transport: Characterization of NadR Ribonucleotide Kinase Mutants ofHaemophilus influenzae

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Abstract
Previously, we characterized a pathway necessary for the processing of NAD+and for uptake of nicotinamide riboside (NR) inHaemophilus 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 anadV+(nicotinamide phosphoribosyltransferase) background, indicating that the ribonucleotide kinase domain is essential for cell viability inH. 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.