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. 2002 Dec;184(24):6906-17.
doi: 10.1128/JB.184.24.6906-6917.2002.

Ribosylnicotinamide kinase domain of NadR protein: identification and implications in NAD biosynthesis

Oleg V Kurnasov  1 , Boris M Polanuyer, Shubha Ananta, Roman Sloutsky, Annie Tam, Svetlana Y Gerdes, Andrei L Osterman
Affiliations

Ribosylnicotinamide kinase domain of NadR protein: identification and implications in NAD biosynthesis

Oleg V Kurnasov et al. J Bacteriol. 2002 Dec.

Erratum in

  • J Bacteriol 2003 Jan;185(2):698

Abstract

NAD is an indispensable redox cofactor in all organisms. Most of the genes required for NAD biosynthesis in various species are known. Ribosylnicotinamide kinase (RNK) was among the few unknown (missing) genes involved with NAD salvage and recycling pathways. Using a comparative genome analysis involving reconstruction of NAD metabolism from genomic data, we predicted and experimentally verified that bacterial RNK is encoded within the 3' region of the nadR gene. Based on these results and previous data, the full-size multifunctional NadR protein (as in Escherichia coli) is composed of (i) an N-terminal DNA-binding domain involved in the transcriptional regulation of NAD biosynthesis, (ii) a central nicotinamide mononucleotide adenylyltransferase (NMNAT) domain, and (iii) a C-terminal RNK domain. The RNK and NMNAT enzymatic activities of recombinant NadR proteins from Salmonella enterica serovar Typhimurium and Haemophilus influenzae were quantitatively characterized. We propose a model for the complete salvage pathway from exogenous N-ribosylnicotinamide to NAD which involves the concerted action of the PnuC transporter and NRK, followed by the NMNAT activity of the NadR protein. Both the pnuC and nadR genes were proven to be essential for the growth and survival of H. influenzae, thus implicating them as potential narrow-spectrum drug targets.

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Figures

FIG. 1.
FIG. 1.
Biochemical transformations and pathways in the biosynthesis of NAD(P) in Enterobacteriaceae and Pasteurellaceae. Pathways and individual enzymatic steps that occur only in E. coli and not in H. influenzae are outlined by dashed boxes and arrows. Common components between E. coli and H. influenzae are shown on a black background. Gene names are given as for E. coli except with nadN (characterized for H. influenzae), hel (characterized for H. influenzae, not present in E. coli), and nadV (characterized for H. ducreyi, not present in either E. coli or H. influenzae). Missing genes are labeled with “(?).”
FIG. 2.
FIG. 2.
Schematic of the N-ribosylnicotinamide salvage pathway of NAD biosynthesis.
FIG.3.
FIG.3.
Gene clustering, domain organization, and amino acid sequence conservation in NadR homologs. (A) Homologous ORFs that clustered in the vicinity of pnuC genes on the chromosomes of several species are outlined by matching patterns. For nadR homologs, the presence of the corresponding domains (HTH, NMNAT, and RNK) is shown. (B) Domain organization and approximate domain boundaries in ecNadR protein. (C) Alignment of multiple amino acid sequences of representative NadR homologs from P. aeruginosa (PA; gi|15597153), M. tuberculosis (MT; gi|15607353), N. punctiforme (NP; http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/framik?taxid = 63737), E. coli (EC; gi|730107), P. multocida (PM; gi|15603252), and H. influenzae (HI; gi|1171638). Conserved amino acids are outlined by black (for identity) and gray (for similarity) backgrounds based on a larger alignment of 15 NadR homologs produced by using ClustalW (http://dot.imgen.bcm.tmc.edu:9331/multi-align/Options/clustalw.html) and Boxshade 3.21 (http://www.isrec.isb-sib.ch:8080/software/BOX_form.html). Conserved sequence motifs characteristic of nucleotidyltransferases (HXGH motif) and P-loop kinases (Walker A and B motifs) are indicated. The N-terminal extension in hiNadR preceding the alternative translation start (Met-53) is shown in lowercase letters.
FIG. 4.
FIG. 4.
Direct verification of NMNAT and RNK activities of hiNadR and stNadR. Reaction products were analyzed by ion-pair HPLC. Shown are the HPLC profiles of the N-ribosylnicotinamide substrate before (A) and after incubation with hiNadR (B) and stNadR (C) and of the NMN substrate before (D) and after incubation with hiNadR (E) and stNadR (F). Substrates and product retention times are shown with dotted vertical lines. Unidentified peaks are marked by “x” and “xx.” ATP and ADP were eluted at higher retention times (data not shown).
FIG. 5.
FIG. 5.
NMNAT steady-state kinetics of hiNadR and stNadR. Apparent kinetic parameters derived at ATP concentrations close to saturation (0.2 mM) are shown for each enzyme. Only an estimate of kinetic parameters could be obtained for stNadR, due to its poor affinity for NMN. V/E, ratio of initial reaction rate (V, in micromoles per second) to enzyme concentration (E, micromolar).
FIG. 6.
FIG. 6.
Genetic footprinting of the H. influenzae chromosomal region containing nadR. (A) Random insertions were introduced into the nadR locus via transposition in vitro and the transformation of H. influenzae. (B) Transformants capable of forming colonies on BHI plates were pooled, and their genomic DNA was isolated and used for insert detection. Detection of insertions was performed by PCR as illustrated. In each PCR, one chromosome-specific primer (FP0, FP1, or RP1) and one transposon (Tn10 or Tn12)-specific primer were used (marked above each lane of the gel image; only three out of six used primer combinations are shown). Each band on the gel originates from a specific transposon insertion, which can thus be mapped in the H. influenzae chromosome (C). Note that areas on the gel that correspond to nadR do not contain any PCR products. This indicates that all insertions in this gene were lethal and were not represented in the analyzed pool of viable mutants. Lane M, molecular size markers. (C) ORFs of the H. influenzae chromosome containing transposon insertions (nonessential) are shown by white arrows, and those without insertions (essential) are shown by black arrows. PCR primers used for the fragment amplification and for insert detection are shown with arrows under the map. Detected transposon insertions are shown with vertical lines above the map.
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