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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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Contact regions between synthetase (blue) and glutaminase (cyan) domains in NADS from Mycobacterium tuberculosis.The majority of interacting residues were found in following structural regions-the α9, α18 helices and the extended C-terminal loop, which are highlighted by pink, green and yellow colors, respectively.
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pone-0039115-g006: Contact regions between synthetase (blue) and glutaminase (cyan) domains in NADS from Mycobacterium tuberculosis.The majority of interacting residues were found in following structural regions-the α9, α18 helices and the extended C-terminal loop, which are highlighted by pink, green and yellow colors, respectively.

Mentions: We hypothesized that structural elements discriminating between the two NADS groups may reside within the looping regions of enzymes from the first group that are responsible for the interactions between the S- and G-domains (or subunits), and that do not have corresponding regions in the single-component enzymes. Thus, all four structural components of an S-domain comprising an interaction interface with the G-domain: the α9, α18 and α20 helices and an extended C-terminal loop (as identified in the original M. tuberculosis NADS structure, Figure 6), appear to play the same role in other reported type F NADS structures from Cytophaga hutchinsonii (PDB:3ILV), and Streptomyces avermitiis (PDB:3N05). The respective regions of the amino acid sequence are conserved in all three branches (I–III) of the phylogenetic tree covering type F enzymes. On the other hand, two of these structural elements, the α18 helix and the C-terminal loop, are absent in all five reported 3D structures from the major branch VI representing type N single-domain enzymes, including st_NADS. A multiple alignment rebuilt to include all variable regions confirms that these structural elements are absent in all representatives of this branch (Table 2 and Figure S2). Of these two elements, the α18 helix is detectable in three reported 3D structures from the branch IV and V. However, none of these structures include a C-terminal loop. The complete multiple alignment suggests that the latter is a signature element distinguishing predicted two-subunit enzymes mapped in these two branches (such as tt_NADS in branch IV) from their single-component counterparts (such as mj_NADS in the predominantly archaeal branch V, see Figure S3).


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)

Contact regions between synthetase (blue) and glutaminase (cyan) domains in NADS from Mycobacterium tuberculosis.The majority of interacting residues were found in following structural regions-the α9, α18 helices and the extended C-terminal loop, which are highlighted by pink, green and yellow colors, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0039115-g006: Contact regions between synthetase (blue) and glutaminase (cyan) domains in NADS from Mycobacterium tuberculosis.The majority of interacting residues were found in following structural regions-the α9, α18 helices and the extended C-terminal loop, which are highlighted by pink, green and yellow colors, respectively.
Mentions: We hypothesized that structural elements discriminating between the two NADS groups may reside within the looping regions of enzymes from the first group that are responsible for the interactions between the S- and G-domains (or subunits), and that do not have corresponding regions in the single-component enzymes. Thus, all four structural components of an S-domain comprising an interaction interface with the G-domain: the α9, α18 and α20 helices and an extended C-terminal loop (as identified in the original M. tuberculosis NADS structure, Figure 6), appear to play the same role in other reported type F NADS structures from Cytophaga hutchinsonii (PDB:3ILV), and Streptomyces avermitiis (PDB:3N05). The respective regions of the amino acid sequence are conserved in all three branches (I–III) of the phylogenetic tree covering type F enzymes. On the other hand, two of these structural elements, the α18 helix and the C-terminal loop, are absent in all five reported 3D structures from the major branch VI representing type N single-domain enzymes, including st_NADS. A multiple alignment rebuilt to include all variable regions confirms that these structural elements are absent in all representatives of this branch (Table 2 and Figure S2). Of these two elements, the α18 helix is detectable in three reported 3D structures from the branch IV and V. However, none of these structures include a C-terminal loop. The complete multiple alignment suggests that the latter is a signature element distinguishing predicted two-subunit enzymes mapped in these two branches (such as tt_NADS in branch IV) from their single-component counterparts (such as mj_NADS in the predominantly archaeal branch V, see Figure S3).

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