Limits...
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.

Show MeSH

Related in: MedlinePlus

Phylogenetic analysis of NAD synthetase enzyme family.(A) Schematic representation of NAD synthetase phylogenetic tree (full version is in Figure S1) constructed based on synthetase domain. Defined types of NAD synthetase genes – “Fused” (type F), “Clustered” (type C), “Remote” (type R) and “None” (type N) are highlighted by red, green, cyan and magenta colors, respectively. The whole tree is partitioned by topology into clusters, which are designated as I–VII branches. (B) Schematic representation of species tree with mapping of NAD synthetase gene types (full version is in Figure S2). Genomes containing single NAD synthetase gene of F, N, C, and R types are depicted by red, green, cyan and magenta colors, respectively. Genomes that possess more than one NAD synthetase gene are divided into “multiple F”, “multiple N”, “single F – single N” and “all others” genome groups, which are highlighted by dark red, dark green, orange and yellow colors, respectively.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3376133&req=5

pone-0039115-g005: Phylogenetic analysis of NAD synthetase enzyme family.(A) Schematic representation of NAD synthetase phylogenetic tree (full version is in Figure S1) constructed based on synthetase domain. Defined types of NAD synthetase genes – “Fused” (type F), “Clustered” (type C), “Remote” (type R) and “None” (type N) are highlighted by red, green, cyan and magenta colors, respectively. The whole tree is partitioned by topology into clusters, which are designated as I–VII branches. (B) Schematic representation of species tree with mapping of NAD synthetase gene types (full version is in Figure S2). Genomes containing single NAD synthetase gene of F, N, C, and R types are depicted by red, green, cyan and magenta colors, respectively. Genomes that possess more than one NAD synthetase gene are divided into “multiple F”, “multiple N”, “single F – single N” and “all others” genome groups, which are highlighted by dark red, dark green, orange and yellow colors, respectively.

Mentions: A projection of the classification described above onto the NADS family phylogenetic tree (Figure 5A, for details see Figure S1) shows remarkable consistency for the three branches I–III covering all enzymes of the type F. While most of NADS sequences within branches IV–VII belong to the type N, representatives of the type C (and R) are found intertwined among them within branches IV and V. Therefore, sequence-based discrimination between genuine single-component (ammonia-utilizing) NADS and two-subunit (glutamine-utilizing) enzymes represents a challenge. It is particularly important since many genomes contain distant representatives of the nitrilase family with yet unassigned functions that could be considered candidate G-subunits for NADS enzymes presently classified as type N.


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)

Phylogenetic analysis of NAD synthetase enzyme family.(A) Schematic representation of NAD synthetase phylogenetic tree (full version is in Figure S1) constructed based on synthetase domain. Defined types of NAD synthetase genes – “Fused” (type F), “Clustered” (type C), “Remote” (type R) and “None” (type N) are highlighted by red, green, cyan and magenta colors, respectively. The whole tree is partitioned by topology into clusters, which are designated as I–VII branches. (B) Schematic representation of species tree with mapping of NAD synthetase gene types (full version is in Figure S2). Genomes containing single NAD synthetase gene of F, N, C, and R types are depicted by red, green, cyan and magenta colors, respectively. Genomes that possess more than one NAD synthetase gene are divided into “multiple F”, “multiple N”, “single F – single N” and “all others” genome groups, which are highlighted by dark red, dark green, orange and yellow colors, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0039115-g005: Phylogenetic analysis of NAD synthetase enzyme family.(A) Schematic representation of NAD synthetase phylogenetic tree (full version is in Figure S1) constructed based on synthetase domain. Defined types of NAD synthetase genes – “Fused” (type F), “Clustered” (type C), “Remote” (type R) and “None” (type N) are highlighted by red, green, cyan and magenta colors, respectively. The whole tree is partitioned by topology into clusters, which are designated as I–VII branches. (B) Schematic representation of species tree with mapping of NAD synthetase gene types (full version is in Figure S2). Genomes containing single NAD synthetase gene of F, N, C, and R types are depicted by red, green, cyan and magenta colors, respectively. Genomes that possess more than one NAD synthetase gene are divided into “multiple F”, “multiple N”, “single F – single N” and “all others” genome groups, which are highlighted by dark red, dark green, orange and yellow colors, respectively.
Mentions: A projection of the classification described above onto the NADS family phylogenetic tree (Figure 5A, for details see Figure S1) shows remarkable consistency for the three branches I–III covering all enzymes of the type F. While most of NADS sequences within branches IV–VII belong to the type N, representatives of the type C (and R) are found intertwined among them within branches IV and V. Therefore, sequence-based discrimination between genuine single-component (ammonia-utilizing) NADS and two-subunit (glutamine-utilizing) enzymes represents a challenge. It is particularly important since many genomes contain distant representatives of the nitrilase family with yet unassigned functions that could be considered candidate G-subunits for NADS enzymes presently classified as type N.

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