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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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Biochemical characterization of T. thermophilus glutaminase and NAD synthetase subunits.SDS-page analysis of Ni-NTA affinity column (A) and gel filtration chromatography (B) elution fractions show that His-tagged recombinant T. thermophilus NADS and untagged GAT tend to co-purify. (C) Kinetic characterization of T. thermophilus S-subunit and G/S complex.
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pone-0039115-g003: Biochemical characterization of T. thermophilus glutaminase and NAD synthetase subunits.SDS-page analysis of Ni-NTA affinity column (A) and gel filtration chromatography (B) elution fractions show that His-tagged recombinant T. thermophilus NADS and untagged GAT tend to co-purify. (C) Kinetic characterization of T. thermophilus S-subunit and G/S complex.

Mentions: The co-expression in E. coli of the two genes forming an operon (TTC1538 – TTC1539) in T. thermophilus showed that their products encoding putative S- and G- subunit of NADS form a tight complex and tend to co-purify on Ni-NTA (Figure 3A) and gel filtration (Figure 3B) chromatography. The SDS-PAGE analysis is suggestive of 1∶1 stoichiometry. The enzymatic characterization of this complex confirmed its NADS activity and the ability to use both, ammonia and glutamine as amide donors. In contrast, the S-subunit alone, when expressed and purified as a single gene, had a comparable NADS activity only with ammonia (Figure 3C) showing Km values very close to typical bacterial NadE enzymes [25], [52]. Notably, even for the two-subunit (G/S) enzyme, the substrate preference was still in favor of ammonia over glutamine (∼50-fold), suggesting a theoretical possibility of using both substrates in vivo. On the other hand, the preference for ammonia over glutamine reported for a typical NadE enzyme (e.g. ∼ 2,500 fold for the NadE from Pseudomonas sp.[25] is substantially higher. In fact, even a low NadE activity observed with glutamine is likely due to its spontaneous hydrolysis and/or some contamination by free ammonia. Overall, the obtained results provided the first experimental verification of the predicted type C (and, possibly, type R) glutamine-utilizing NADS enzymes (Figure 2).


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)

Biochemical characterization of T. thermophilus glutaminase and NAD synthetase subunits.SDS-page analysis of Ni-NTA affinity column (A) and gel filtration chromatography (B) elution fractions show that His-tagged recombinant T. thermophilus NADS and untagged GAT tend to co-purify. (C) Kinetic characterization of T. thermophilus S-subunit and G/S complex.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0039115-g003: Biochemical characterization of T. thermophilus glutaminase and NAD synthetase subunits.SDS-page analysis of Ni-NTA affinity column (A) and gel filtration chromatography (B) elution fractions show that His-tagged recombinant T. thermophilus NADS and untagged GAT tend to co-purify. (C) Kinetic characterization of T. thermophilus S-subunit and G/S complex.
Mentions: The co-expression in E. coli of the two genes forming an operon (TTC1538 – TTC1539) in T. thermophilus showed that their products encoding putative S- and G- subunit of NADS form a tight complex and tend to co-purify on Ni-NTA (Figure 3A) and gel filtration (Figure 3B) chromatography. The SDS-PAGE analysis is suggestive of 1∶1 stoichiometry. The enzymatic characterization of this complex confirmed its NADS activity and the ability to use both, ammonia and glutamine as amide donors. In contrast, the S-subunit alone, when expressed and purified as a single gene, had a comparable NADS activity only with ammonia (Figure 3C) showing Km values very close to typical bacterial NadE enzymes [25], [52]. Notably, even for the two-subunit (G/S) enzyme, the substrate preference was still in favor of ammonia over glutamine (∼50-fold), suggesting a theoretical possibility of using both substrates in vivo. On the other hand, the preference for ammonia over glutamine reported for a typical NadE enzyme (e.g. ∼ 2,500 fold for the NadE from Pseudomonas sp.[25] is substantially higher. In fact, even a low NadE activity observed with glutamine is likely due to its spontaneous hydrolysis and/or some contamination by free ammonia. Overall, the obtained results provided the first experimental verification of the predicted type C (and, possibly, type R) glutamine-utilizing NADS enzymes (Figure 2).

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