NAD+ biosynthesis in bacteria is controlled by global carbon/nitrogen levels via PII signaling

Abstract

Nicotinamideadenine dinucleotide (NAD + ) is a central metabolite participating in core metabolic redox reactions. The prokaryotic NAD synthetase enzyme NadE catalyzes the last step of NAD + biosynthesis, converting nicotinic acid adenine dinucleotide (NaAD) to NAD + . Some members of the NadE family use L-glutamine as a nitrogen donor and are named NadE Gln . Previous gene neighborhood analysis has indicated that the bacterial nadE gene is frequently clustered with the gene encoding the regulatory signal transduction protein PII, suggesting a functional relationship between these proteins in response to the nutritional status and the carbon/nitrogen ratio of the bacterial cell. Here, using affinity chromatography, bioinformatics analyses, NAD synthetase activity and biolayer interferometry assays, we show that PII and NadE Gln physically interact in vitro, that this complex relieves NadE Gln negative feedback inhibition by NAD + . This mechanism is conserved in distantly related bacteria. Of note, the PII protein allosteric effector and cellular nitrogen level indicator 2-oxoglutarate (2-OG) inhibited the formation of the PII-NadE Gln complex within a physiological range. These results indicate an interplay between the levels of ATP, ADP, 2-OG, PII-sensed glutamine, and NAD + , representing a metabolic hub that may balance the levels of core nitrogen and carbon metabolites. Our findings support the notion that PII proteins act as a dissociable regulatory subunit of NadE Gln , thereby enabling the control of NAD + biosynthesis according to the nutritional status of the bacterial cell. https://www.jbc.org/cgi/

The novel P II -interactor PirC identifies phosphoglycerate mutase as key control point of carbon storage metabolism in cyanobacteria

“…A variety of key metabolic enzymes, transcription factors, and transport proteins use this signaling path to tune their activity in response to the metabolic state. P II in its different conformations can directly interact with various target proteins such as the N-acetyl- l -glutamate kinase, catalyzing the committed step in arginine biosynthesis ( 7 , 8 ); the acetyl-CoA carboxylase, catalyzing the rate-limiting step in fatty acid biosynthesis ( 9 ); the phosphoenolpyruvate carboxylase, which catalyzes an anaplerotic carbon fixation ( 10 ); or the glutamine-dependent nicotinamide adenine dinucleotide (NAD + ) synthetase ( 11 ). Besides tuning the activity of enzymes, recent analyses revealed that, through direct protein–protein interaction, the abundant P II proteins can also regulate transport activities, including an ensemble of nitrogen transporters such as the nitrate/nitrite transport system, the urea transport system, and the ammonium transporter ( 12 ).…”

“…In a previous P II protein interaction study, several putative P II interactors of unknown function were identified ( 12 ). The most prominent hit was the product of the sll0944 gene, a member of the NtcA regulon ( 11 , 13 ). The sll0944 gene product is annotated in Uniprot ( https://www.uniprot.org/uniprot/P77971 ) as a protein of unknown function.…”

The Protein-Protein Interaction Network Reveals a Novel Role of the Signal Transduction Protein PII in the Control of c-di-GMP Homeostasis in Azospirillum brasilense

“…Two isoforms of the glutamine-dependent NAD synthetase (NadE1 and NadE2) and the enzyme involved in coenzyme A biosynthesis (CoaBC) were identified as potential GlnZ targets ( Table 1 ). The NadE2 enzyme has been studied as a GlnZ target in A. brasilense previously ( 26 ). Upon an ammonium shock, GlnZ interacts with NadE2 to relieve the NAD + negative-feedback inhibition over the NadE2 enzyme.…”

“…Recent work in bacteria and plants revealed several novel PII targets that are not only involved in nitrogen metabolism, but in general core metabolism, such as acetyl-coenzyme A (CoA) carboxylase, phosphoenolpyruvate carboxylase (PEPC), NAD synthetase, and glucosamine 6-phosphate deaminase ( 25 – 27 ). Therefore, PII has a much broader regulatory role than originally thought.…”

“…Therefore, PII has a much broader regulatory role than originally thought. We have previously used PII affinity columns to successfully identify acetyl-CoA carboxylase and NAD synthetase as binding partners of the PII protein GlnZ in A. brasilense ( 26 , 28 ). Here, we performed multiple GlnZ ligand fishing assays to identify 37 proteins that are likely to be part of the PII protein interaction network in A. brasilense .…”