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

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.