Abstract
NAD is a ubiquitous and essential metabolic redox cofactor which also functions as a substrate in certain regulatory pathways. The last step of NAD synthesis is the ATP-dependent amidation of deamido-NAD by NAD synthetase (NADS). Members of the NADS family are present in nearly all species across the three kingdoms of Life. In eukaryotic NADS, the core synthetase domain is fused with a nitrilase-like glutaminase domain supplying ammonia for the reaction. This two-domain NADS arrangement enabling the utilization of glutamine as nitrogen donor is also present in various bacterial lineages. However, many other bacterial members of NADS family do not contain a glutaminase domain, and they can utilize only ammonia (but not glutamine) in vitro. A single-domain NADS is also characteristic for nearly all Archaea, and its dependence on ammonia was demonstrated here for the representative enzyme from Methanocaldococcus jannaschi. However, a question about the actual in vivo nitrogen donor for single-domain members of the NADS family remained open: Is it glutamine hydrolyzed by a committed (but yet unknown) glutaminase subunit, as in most ATP-dependent amidotransferases, or free ammonia as in glutamine synthetase? Here we addressed this dilemma by combining evolutionary analysis of the NADS family with experimental characterization of two representative bacterial systems: a two-subunit NADS from Thermus thermophilus and a single-domain NADS from Salmonella typhimurium providing evidence that ammonia (and not glutamine) is the physiological substrate of a typical single-domain NADS. The latter represents the most likely ancestral form of NADS. The ability to utilize glutamine appears to have evolved via recruitment of a glutaminase subunit followed by domain fusion in an early branch of Bacteria. Further evolution of the NADS family included lineage-specific loss of one of the two alternative forms and horizontal gene transfer events. Lastly, we identified NADS structural elements associated with glutamine-utilizing capabilities.
Highlights
Nicotinamide adenine dinucleotide (NAD) serves both, as a ubiquitous cofactor in hundreds of redox reactions and as a substrate in a number of regulatory processes related to cell cycle and longevity, calcium signaling, immune response, DNA repair, etc. [1,2,3]
NAD synthetase (NADS) was demonstrated to be essential in a number of bacterial pathogens including Mycobacterium tuberculosis, Bacillus anthracis, Staphylococcus aureus, and Escherichia coli [5,6], and it is currently being pursued as a target for antibiotic development [7,8]
NADS family phylogenomic distribution and classification Among, 940 complete genomes included in the manually curated metabolic subsystem ‘‘NAD and NADP metabolism’’ [49] in the SEED database [39], at least one form of NADS was found to be present in 886 genomes (94%) including 814 bacterial, 56 archaeal and 16 eukaryotics genomes (Table S3)
Summary
Nicotinamide adenine dinucleotide (NAD) serves both, as a ubiquitous cofactor in hundreds of redox reactions and as a substrate in a number of regulatory processes related to cell cycle and longevity, calcium signaling, immune response, DNA repair, etc. [1,2,3]. Due to its impact on most aspects of metabolism, NAD is essential for survival and several enzymes involved in its biosynthesis have been recognized as potential drug targets [4,5] One of these enzymes is NAD synthetase (NADS), which catalyzes amidation of nicotinic acid adenine dinucleotide (NaAD) in the last step of NAD synthesis. A pyridine carboxylate group is activated by adenylation followed by amidation via the nucleophilic replacement of the adenylate moiety with ammonia in the second step (Figure 1) This general mechanism involving an adenylation step is shared by other ATP-dependent amidotransferase including GMP synthetase (GuaA), asparagine synthetase B (AsnB) and Glu-tRNAGln amidotransferase (GatABC).
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