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Glutamine versus ammonia utilization in the NAD synthetase family.

De Ingeniis J, Kazanov MD, Shatalin K, Gelfand MS, Osterman AL, Sorci L - PLoS ONE (2012)

Bottom Line: NAD is a ubiquitous and essential metabolic redox cofactor which also functions as a substrate in certain regulatory pathways.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.Lastly, we identified NADS structural elements associated with glutamine-utilizing capabilities.

View Article: PubMed Central - PubMed

Affiliation: Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.

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.

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Scheme of the two-step reaction catalyzed by NAD synthetase.
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pone-0039115-g001: Scheme of the two-step reaction catalyzed by NAD synthetase.

Mentions: NADS, a member of the N-type ATP pyrophosphatase family [14], catalyzes the ATP-dependent transformation of nicotinic acid adenine dinucleotide (NaAD) into the amide product NAD via a two-step process. In the first step, 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). Other amidotransferase such as carbamoylphosphate synthetase (CarAB) and formylglycinamidine ribonucleotide amidotransferase (PurL) – both belonging to the ATP-grasp superfamily – and CTP synthetase (PyrG) – belonging to the P-loop NTPase family – also use ATP in their catalytic mechanism, which apparently includes the hydrolysis of ATP to ADP rather than adenylation followed by AMP release [15] (see Table 1). Another mechanistic feature common for enzymes of this class is the in situ formation of ammonia through deamidation of glutamine to glutamate by a committed glutaminase domain (or subunit). The molecule of ammonia is directly channeled from the glutaminase domain to the amidation site in the synthetase domain (we will further refer to them as G-domain and S-domain, respectively) without dissociation to the milieu. A compact two-domain arrangement allows these enzymes to utilize glutamine in vivo (whereas in vitro they can use both, glutamine and ammonia). This ability is of utmost physiological importance, as the cellular level of free ammonia is typically quite low due to its efficient capturing by glutamine synthetase. The latter enzyme was historically considered as the only ATP-dependent amidotransferase that utilizes ammonia (and not glutamine) in vivo[16].


Glutamine versus ammonia utilization in the NAD synthetase family.

De Ingeniis J, Kazanov MD, Shatalin K, Gelfand MS, Osterman AL, Sorci L - PLoS ONE (2012)

Scheme of the two-step reaction catalyzed by NAD synthetase.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376133&req=5

pone-0039115-g001: Scheme of the two-step reaction catalyzed by NAD synthetase.
Mentions: NADS, a member of the N-type ATP pyrophosphatase family [14], catalyzes the ATP-dependent transformation of nicotinic acid adenine dinucleotide (NaAD) into the amide product NAD via a two-step process. In the first step, 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). Other amidotransferase such as carbamoylphosphate synthetase (CarAB) and formylglycinamidine ribonucleotide amidotransferase (PurL) – both belonging to the ATP-grasp superfamily – and CTP synthetase (PyrG) – belonging to the P-loop NTPase family – also use ATP in their catalytic mechanism, which apparently includes the hydrolysis of ATP to ADP rather than adenylation followed by AMP release [15] (see Table 1). Another mechanistic feature common for enzymes of this class is the in situ formation of ammonia through deamidation of glutamine to glutamate by a committed glutaminase domain (or subunit). The molecule of ammonia is directly channeled from the glutaminase domain to the amidation site in the synthetase domain (we will further refer to them as G-domain and S-domain, respectively) without dissociation to the milieu. A compact two-domain arrangement allows these enzymes to utilize glutamine in vivo (whereas in vitro they can use both, glutamine and ammonia). This ability is of utmost physiological importance, as the cellular level of free ammonia is typically quite low due to its efficient capturing by glutamine synthetase. The latter enzyme was historically considered as the only ATP-dependent amidotransferase that utilizes ammonia (and not glutamine) in vivo[16].

Bottom Line: NAD is a ubiquitous and essential metabolic redox cofactor which also functions as a substrate in certain regulatory pathways.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.Lastly, we identified NADS structural elements associated with glutamine-utilizing capabilities.

View Article: PubMed Central - PubMed

Affiliation: Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.

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.

Show MeSH
Related in: MedlinePlus