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

Abstract

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.

Biosynthesis and Recycling of Nicotinamide Cofactors in Mycobacterium tuberculosis

“…NAD can be degraded by a variety of enzymes, including NAD glycohydrolase, DNA ligase, NAD pyrophosphatase, NAD(P) ϩ nucleosidase, poly(ADP-ribose) polymerase, mono-ADP-ribosyltransferase, and NAD pyrophosphatase (13). In an alternative nondeamidating salvage pathway, nicotinamide phosphoribosyltransferase (encoded by nadV) salvages nicotinamide directly with the resulting NMN subsequently converted to NAD by the NMN adenylyltransferase activity of NadR (17). A third recycling pathway includes the conversion of exogenously scavenged pyridine nucleotides to NAD (17,18).…”

NAD+Utilization inPasteurellaceae: Simplification of a Complex Pathway

“…⌬NRK (NR-kinase deficient) background (48), indicating that NR phosphorylation is required for NR uptake. NRK is the subject for feedback inhibition by the end product NAD ϩ ; hence, NR uptake and NAD ϩ synthesis are feedback regulated by NAD ϩ (48).…”

“…NRK is the subject for feedback inhibition by the end product NAD ϩ ; hence, NR uptake and NAD ϩ synthesis are feedback regulated by NAD ϩ (48). Proposed "open" and "closed" channel forms for PnuC which might respond and interact with NadR are indicated.…”

“…However, whether PnuC represents the primary active carrier system needs further analysis (75). Evidence for the involvement of a second protein function necessary for NR uptake in H. influenzae came from the observation that mutants with mutations in nadR were defective in NR transport (48) and that pnuC mutants could be created, implying a secondary uptake route (35). In Salmonella, the NadR protein acts as a repressor able to control the transcription of the nadB, nadA-pnuC, and pncB genes, depending on cellular NAD ϩ levels (64,65).…”

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

“…The enzymatic activities of NadR are the key players that connect transport mediated by PnuC to NAD metabolism. The NR kinase domain indirectly regulates the rate of NR uptake mediated by PnuC (Grose et al, 2005a,b;Merdanovic et al, 2005). The phosphorylation of NR to NMN subsequent to its transport prevents efflux of the substrate, thus creating directionality and providing a possibility of substrate accumulation within the cell (Kurnasov et al, 2002) (Figure 5).…”