Regulation of NAD metabolism in Salmonella typhimurium: Genetic analysis and cloning of the nadR repressor locus

Foster, John W.; Holley-Guthrie, Elizabeth; Warren, Felicity

doi:10.1007/bf00330454

Cited by 23 publications

(21 citation statements)

References 23 publications

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“…Although we cannot rule out the possibility that binding of the non-active-site NAD molecule is a crystallization artifact, the specific interactions between this NAD molecule and hiNadR tetramer indicate that it may have biological implications. In E. coli and S. typhimurium, the NadR protein represses three genes involved in NAD biosynthesis in a NAD-dependent manner (15,16). It is possible that the NAD molecule as an effector may bind to a site different from the active site of NMNAT domain.…”

Section: Resultsmentioning

confidence: 99%

“…2 In addition to the NMNAT activity, NadR in S. typhimurium and E. coli contain a DNA-binding helix-turn-helix motif N-terminal to the NMNAT domain. The S. typhimurium NadR has long been demonstrated to be a NAD-dependent repressor for the transcription of genes involved in both de novo biosynthesis (nadB and nadA) and niacin salvage (pncB) (15,16). It was also suggested that NadR may interact with an integral membrane transporter PnuC protein and directly participate in the uptake of exogenous NAD precursors (17).…”

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confidence: 99%

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Crystal Structure of Haemophilus influenzae NadR Protein

Singh

Kurnasov

Chen

et al. 2002

Journal of Biological Chemistry

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Haemophilus influenzae NadR protein (hiNadR) has been shown to be a bifunctional enzyme possessing both NMN adenylytransferase (NMNAT; EC 2.7.7.1) and ribosylnicotinamide kinase (RNK; EC 2.7.1.22) activities. Its function is essential for the growth and survival of H. influenzae and thus may present a new highly specific anti-infectious drug target. We have solved the crystal structure of hiNadR complexed with NAD using the selenomethionine MAD phasing method. The structure reveals the presence of two distinct domains. The N-terminal domain that hosts the NMNAT activity is closely related to archaeal NMNAT, whereas the C-terminal domain, which has been experimentally demonstrated to possess ribosylnicotinamide kinase activity, is structurally similar to yeast thymidylate kinase and several other P-loop-containing kinases. There appears to be no cross-talk between the two active sites. The bound NAD at the active site of the NMNAT domain reveals several critical interactions between NAD and the protein. There is also a second non-active-site NAD molecule associated with the C-terminal RNK domain that adopts a highly folded conformation with the nicotinamide ring stacking over the adenine base. Whereas the RNK domain of the hiNadR structure presented here is the first structural characterization of a ribosylnicotinamide kinase from any organism, the NMNAT domain of hiNadR defines yet another member of the pyridine nucleotide adenylyltransferase family.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Crystal Structure of Haemophilus influenzae NadR Protein

Singh

Kurnasov

Chen

et al. 2002

Journal of Biological Chemistry

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“…The initial steps of the de novo biosynthetic pathway, L-aspartate oxidase and quinolinic acid synthetase, and one of the components of the scavenging system, nicotinic acid (NA) phosphoribosyltransferase (pncB), are transcriptionally regulated in Salmonella typhimurium as determined by lacZ operon fusion studies. Genes encoding quinolinic acid synthetase (nadA) and L-aspartate oxidase (nadB) are tnore tightly controlled than the pncB locus (4,8,9,17,18). However, all three loci are under negative transcriptional control by the product of the nadR regulatory locus (4,8).…”

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confidence: 99%

“…Genes encoding quinolinic acid synthetase (nadA) and L-aspartate oxidase (nadB) are tnore tightly controlled than the pncB locus (4,8,9,17,18). However, all three loci are under negative transcriptional control by the product of the nadR regulatory locus (4,8). The nadR locus is located at 99 min on the Salmonella linkage map.…”

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confidence: 99%

Regulation of NAD metabolism in Salmonella typhimurium: molecular sequence analysis of the bifunctional nadR regulator and the nadA-pnuC operon

et al. 1990

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In Salmonella typhimurium, de novo synthesis of NAD is regulated through the transcriptional control of the nadA and nadB loci. Likewise, the pyridine nucleotide salvage pathway is controlled at pncB. The transcriptional expression of these three loci is coordinately regulated by the product of nadR. However, there is genetic evidence suggesting that NadR is bifunctional, serving in both regulatory and transport capacities. One class of mutations in the nadR locus imparts a transport-defective PnuA-phenotype. These mutants retain regulation properties but are unable to transport nicotinamide mononucleotide (NMN) intact across the cell membrane. Other nadR mutants lose both regulatory and transport capabilities, while a third class loses only regulatory ability. The unusual NMN transport activity requires both the PnuC and NadR proteins, with the pnuC locus residing in an operon with nadA. To prove that nadlR encoded a single protein and to gain insight into a regulatory target locus, the nadR and nadA pnuC loci were cloned and sequenced. A DNA fragment which complemented both regulatory and transport mutations was found to contain a single open reading frame capable of encoding a 409-amino-acid protein (47,022 daltons), indicating that NadR is indeed bifunctional. Confirmation of the operon arrangement for nadA and pnuC was obtained through the sequence analysis of a 2.4-kilobase DNA fragment which complemented both NadA and PnuC mutant phenotypes. The nadA product, confirmed in maxicells, was a 365-amino-acid protein (40,759 daltons), while pnuC encoded a 322-amino-acid protein (36,930 daltons). The extremely hydrophobic (71%) nature of the PnuC protein indicated that it was an integral membrane protein, consistent with its central role in the transport of NMN across the cytoplasmic membrane. The results presented here and in previous studies suggest a hypothetical model in which NadR interacts with PnuC at low internal NAD levels, permitting transport of NMN intact into the cell. As NAD levels increase within the cell, the affinity of NadR for the operator regions of nadA, nadB, and pncB increases, repressing the transcription of these target genes.

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“…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. NadR was initially thought to have a regulatory as well as a transport function, because it is able to sense the internal NAD pool in order to repress essential genes for NAD biosynthesis [nadB, nadA-pnuC, and pncB (Spector et al, 1985;Cookson et al, 1987;Zhu et al, 1989;Penfound and Foster, 1999)] and to regulate transport via PnuC in response to cellular NAD levels (Tirgari et al, 1986;Foster et al, 1990;Foster and Penfound, 1993).…”

Section: A General Scheme For Pnu Transportermediated Uptakementioning

confidence: 99%

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

Jaehme

Slotboom

2015

Biological Chemistry

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Many bacteria can take up vitamins from the environment via specific transport machineries. Uptake is essential for organisms that lack complete vitamin biosynthesis pathways, but even in the presence of biosynthesis routes uptake is likely preferred, because it is energetically less costly. Pnu transporters represent a class of membrane transporters for a diverse set of B-type vitamins. They were identified 30 years ago and catalyze transport by the mechanism of facilitated diffusion, without direct coupling to ATP hydrolysis or transport of coupling ions. Instead, directionality is achieved by metabolic trapping, in which the vitamin substrate is converted into a derivative that cannot be transported, for instance by phosphorylation. The recent crystal structure of the nicotinamide riboside transporter PnuC has provided the first insights in substrate recognition and selectivity. Here, we will summarize the current knowledge about the function, structure, and evolution of Pnu transporters. Additionally, we will highlight their role for potential biotechnological and pharmaceutical applications.

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Regulation of NAD metabolism in Salmonella typhimurium: Genetic analysis and cloning of the nadR repressor locus

Cited by 23 publications

References 23 publications

Crystal Structure of Haemophilus influenzae NadR Protein

Crystal Structure of Haemophilus influenzae NadR Protein

Regulation of NAD metabolism in Salmonella typhimurium: molecular sequence analysis of the bifunctional nadR regulator and the nadA-pnuC operon

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

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