Analysis of Escherichia coli nicotinate mononucleotide adenylyltransferase mutants in vivo and in vitro

Stancek, Martin; Schnell, R.; Rydén-Aulin, Monica

doi:10.1186/1471-2091-6-16

Cited by 11 publications

(5 citation statements)

References 24 publications

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“…Phylogenic analysis of PfNMNAT compared to representative prokaryotic and eukaryotic NMNATs demonstrates its similarity to bacterial versions of the enzyme ( Figure 3B ) [31] . Based on its high level of shared identity with the E. coli NMNAT (NadD) (Figure S5 in File S1 ), we tested whether PfNMNAT could genetically complement the essential bacterial enzyme in vivo [32] . We generated an E. coli strain where the nadD locus was replaced with the chloramphenicol (cam) drug resistance cassette ( nadD ::cam) and the non-essential linked gene, ybeT , was replaced with a kanamycin (kan) drug resistance cassette ( ybeT ::kan).…”

Section: Resultsmentioning

confidence: 99%

Targeting NAD+ Metabolism in the Human Malaria Parasite Plasmodium falciparum

et al. 2014

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Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite utilized as a redox cofactor and enzyme substrate in numerous cellular processes. Elevated NAD+ levels have been observed in red blood cells infected with the malaria parasite Plasmodium falciparum, but little is known regarding how the parasite generates NAD+. Here, we employed a mass spectrometry-based metabolomic approach to confirm that P. falciparum lacks the ability to synthesize NAD+ de novo and is reliant on the uptake of exogenous niacin. We characterized several enzymes in the NAD+ pathway and demonstrate cytoplasmic localization for all except the parasite nicotinamidase, which concentrates in the nucleus. One of these enzymes, the P. falciparum nicotinate mononucleotide adenylyltransferase (PfNMNAT), is essential for NAD+ metabolism and is highly diverged from the human homolog, but genetically similar to bacterial NMNATs. Our results demonstrate the enzymatic activity of PfNMNAT in vitro and demonstrate its ability to genetically complement the closely related Escherichia coli NMNAT. Due to the similarity of PfNMNAT to the bacterial enzyme, we tested a panel of previously identified bacterial NMNAT inhibitors and synthesized and screened twenty new derivatives, which demonstrate a range of potency against live parasite culture. These results highlight the importance of the parasite NAD+ metabolic pathway and provide both novel therapeutic targets and promising lead antimalarial compounds.

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Section: Resultsmentioning

confidence: 99%

Targeting NAD+ Metabolism in the Human Malaria Parasite Plasmodium falciparum

et al. 2014

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“…Nicotinamide adenine dinucleotide (NAD + ) is synthesized in cyanobacteria from L-aspartate by a five-step pathway encoded by most bacterial species ( Figure 6B) [106]. The last two enzymes in the pathway, NadD and NadE, have low sequence similarity to the equivalent E. coli proteins but the activity of the enzymes has been confirmed in Synechocystis [107].…”

Section: Nad + and Nadp + Biosynthesismentioning

confidence: 99%

Current knowledge and recent advances in understanding metabolism of the model cyanobacteriumSynechocystissp. PCC 6803

2020

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Cyanobacteria are key organisms in the global ecosystem, useful models for studying metabolic and physiological processes conserved in photosynthetic organisms, and potential renewable platforms for production of chemicals. Characterizing cyanobacterial metabolism and physiology is key to understanding their role in the environment and unlocking their potential for biotechnology applications. Many aspects of cyanobacterial biology differ from heterotrophic bacteria. For example, most cyanobacteria incorporate a series of internal thylakoid membranes where both oxygenic photosynthesis and respiration occur, while CO2 fixation takes place in specialized compartments termed carboxysomes. In this review, we provide a comprehensive summary of our knowledge on cyanobacterial physiology and the pathways in Synechocystis sp. PCC 6803 (Synechocystis) involved in biosynthesis of sugar-based metabolites, amino acids, nucleotides, lipids, cofactors, vitamins, isoprenoids, pigments and cell wall components, in addition to the proteins involved in metabolite transport. While some pathways are conserved between model cyanobacteria, such as Synechocystis, and model heterotrophic bacteria like Escherichia coli, many enzymes and/or pathways involved in the biosynthesis of key metabolites in cyanobacteria have not been completely characterized. These include pathways required for biosynthesis of chorismate and membrane lipids, nucleotides, several amino acids, vitamins and cofactors, and isoprenoids such as plastoquinone, carotenoids, and tocopherols. Moreover, our understanding of photorespiration, lipopolysaccharide assembly and transport, and degradation of lipids, sucrose, most vitamins and amino acids, and haem, is incomplete. We discuss tools that may aid our understanding of cyanobacterial metabolism, notably CyanoSource, a barcoded library of targeted Synechocystis mutants, which will significantly accelerate characterization of individual proteins.

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“…It is present in the vast majority of bacteria, including pathogens, and is essential for cell survival and viability, as experimentally demonstrated in E. coli [71], B. subtilis [72], S. aureus [73], S. typhimurium [74,75], and the cyanobacterium Synechocystis sp. [76]; its selective inhibition might thus represent a breakthrough in the development of novel broad-spectrum antibiotics.…”

Section: Eubacterial Namn Adenylyltransferase (Nadd)mentioning

confidence: 99%

NAD(P) Biosynthesis Enzymes as Potential Targets for Selective Drug Design

Magni

Stefano²,

Orsomando

et al. 2009

CMC

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NAD(P) biosynthetic pathways can be considered a generous source of enzymatic targets for drug development. Key reactions for NAD(P) biosynthesis in all organisms, common to both de novo and salvage routes, are catalyzed by NMN/NaMN adenylyltransferase (NMNAT), NAD synthetase (NADS), and NAD kinase (NADK). These reactions represent a three-step pathway, present in the vast majority of living organisms, which is responsible for the generation of both NAD and NADP cellular pools. The validation of these enzymes as drug targets is based on their essentiality and conservation among a large variety of pathogenic microorganisms, as well as on their differential structural features or their differential metabolic contribution to NAD(P) homeostasis between microbial and human cell types. This review describes the structural and functional properties of eubacterial and human enzymes endowed with NMNAT, NADS, and NADK activities, as well as with nicotinamide phosphoribosyltransferase (NamPRT) and nicotinamide riboside kinase (NRK) activities, highlighting the species-related differences, with emphasis on their relevance for drug design. In addition, since the overall NMNAT activity in humans is accounted by multiple isozymes differentially involved in the metabolic activation of antineoplastic compounds, their individual diagnostic value for early therapy optimization is outlined. The involvement of human NMNAT in neurodegenerative disorders and its role in neuroprotection is also discussed.

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Analysis of Escherichia coli nicotinate mononucleotide adenylyltransferase mutants in vivo and in vitro

Cited by 11 publications

References 24 publications

Targeting NAD+ Metabolism in the Human Malaria Parasite Plasmodium falciparum

Targeting NAD+ Metabolism in the Human Malaria Parasite Plasmodium falciparum

Current knowledge and recent advances in understanding metabolism of the model cyanobacteriumSynechocystissp. PCC 6803

NAD(P) Biosynthesis Enzymes as Potential Targets for Selective Drug Design

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