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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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Analysis of Nit11 and SK51 S. typhimurium mutant strains.(A) Schematic of S. typhimurium nadE mutant strains. Nit11 mutant features a missense mutation at nucleotide 143 yielding the amino acid substitution AN. SK51 mutant features a single nucleotide deletion (G at position – 51 considering as +1 the first base of the start codon) that is just upstream of the -35 box regulatory region of the promoter. Predicted regulatory sequences are indicated in the bottom line. (B) Relative expression level of nadE in the wild type and in the two classes of mutants nit11 (S48N) and SK51 in four different growth conditions: rich medium (1), minimal medium supplemented with 20 mM NH3 (2), MM supplemented with 5 mM (3) or 20 mM (4) glutamine. (C) Kinetic characterization of wild type and S48N S. typhimurium NAD synthetase. Initial rates were measured by spectrophotometrical coupled (SPEC) assays. The kinetic parameters Km and kcat are apparent values determined at fixed (saturating) concentrations of co-substrates. For fixed substrates, concentrations were: 2 mM ATP, 2 mM NaAD, and 40 mM NH3. Errors represent standard deviation.
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pone-0039115-g004: Analysis of Nit11 and SK51 S. typhimurium mutant strains.(A) Schematic of S. typhimurium nadE mutant strains. Nit11 mutant features a missense mutation at nucleotide 143 yielding the amino acid substitution AN. SK51 mutant features a single nucleotide deletion (G at position – 51 considering as +1 the first base of the start codon) that is just upstream of the -35 box regulatory region of the promoter. Predicted regulatory sequences are indicated in the bottom line. (B) Relative expression level of nadE in the wild type and in the two classes of mutants nit11 (S48N) and SK51 in four different growth conditions: rich medium (1), minimal medium supplemented with 20 mM NH3 (2), MM supplemented with 5 mM (3) or 20 mM (4) glutamine. (C) Kinetic characterization of wild type and S48N S. typhimurium NAD synthetase. Initial rates were measured by spectrophotometrical coupled (SPEC) assays. The kinetic parameters Km and kcat are apparent values determined at fixed (saturating) concentrations of co-substrates. For fixed substrates, concentrations were: 2 mM ATP, 2 mM NaAD, and 40 mM NH3. Errors represent standard deviation.

Mentions: Salmonella nit mutants that were previously characterized as defective in nitrogen assimilation despite normal levels of ammonia assimilatory enzymes [36], provided us with a valuable case study to further address the question of the in vivo amide donor characteristic of NadE subfamily. Whereas both classes of mutants described in the original study, those induced by ICR (SK51) or by nitrosoguanidine (nit11), were genetically mapped within the nadE locus, the exact nature of these mutations has not been established. Amplification and sequencing of the respective mutant loci revealed two types of genetic lesions: (i) a deletion of a single nucleotide (G) in the promoter region upstream of the predicted -35 box, and (ii) a point mutation at the nucleotide 143 (G to A) replacing Ser-84 residue with Asn (Figure 4A). Therefore, a reported loss of the NADS activity could be expected to occur at the transcriptional level for the first type, and at the level of enzymatic properties for the second type of mutations. Indeed, a quantitative RT-PCR confirmed a substantial (∼ 100-fold) drop of the nadE mRNA level in SK51 compared to nit11 or wild-type S. typhimurium (Figure 4B). Importantly, increasing ammonia or glutamine in the media did not have any effect on the relative level of the nadE mRNA. It suggests that the observed suppression of growth phenotype is not due to the regulation on the level of transcription, but rather due to overcoming a competition for nitrogen source between a severely suppressed NADS and a robust glutamine synthetase (GlnA). The latter interpretation is consistent with the reported compensatory effect of the glnA mutation [36]. To determine the effect of the mutation nit11 on the enzyme function, wild type st_NADS and S48N mutant were overexpressed in E. coli, purified and their apparent steady-state kinetic parameters were determined toward ATP, NaAD, and NH3 (Figure 4C). The wild-type enzyme had kinetic parameters very close to those reported for E. coli NadE [52]. The most dramatic difference from S48N mutant was observed at the level of the apparent Km value for ammonia, which was ∼ 70-fold higher for the mutant st_NADS compared to the wild-type enzyme. This finding provides a straightforward interpretation for the observed growth phenotype of nit11 strain, which could be compensated for by increasing the concentration of ammonia in the media [36]. Notably, the mutated residue is a part of the conserved motif SGGXDST characteristic of the N-type ATP pyrophosphatase family [14]. Based on the NadE structure [34], it is located in the vicinity of the active site, which is consistent with the observed effect on the NADS activity.


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)

Analysis of Nit11 and SK51 S. typhimurium mutant strains.(A) Schematic of S. typhimurium nadE mutant strains. Nit11 mutant features a missense mutation at nucleotide 143 yielding the amino acid substitution AN. SK51 mutant features a single nucleotide deletion (G at position – 51 considering as +1 the first base of the start codon) that is just upstream of the -35 box regulatory region of the promoter. Predicted regulatory sequences are indicated in the bottom line. (B) Relative expression level of nadE in the wild type and in the two classes of mutants nit11 (S48N) and SK51 in four different growth conditions: rich medium (1), minimal medium supplemented with 20 mM NH3 (2), MM supplemented with 5 mM (3) or 20 mM (4) glutamine. (C) Kinetic characterization of wild type and S48N S. typhimurium NAD synthetase. Initial rates were measured by spectrophotometrical coupled (SPEC) assays. The kinetic parameters Km and kcat are apparent values determined at fixed (saturating) concentrations of co-substrates. For fixed substrates, concentrations were: 2 mM ATP, 2 mM NaAD, and 40 mM NH3. Errors represent standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0039115-g004: Analysis of Nit11 and SK51 S. typhimurium mutant strains.(A) Schematic of S. typhimurium nadE mutant strains. Nit11 mutant features a missense mutation at nucleotide 143 yielding the amino acid substitution AN. SK51 mutant features a single nucleotide deletion (G at position – 51 considering as +1 the first base of the start codon) that is just upstream of the -35 box regulatory region of the promoter. Predicted regulatory sequences are indicated in the bottom line. (B) Relative expression level of nadE in the wild type and in the two classes of mutants nit11 (S48N) and SK51 in four different growth conditions: rich medium (1), minimal medium supplemented with 20 mM NH3 (2), MM supplemented with 5 mM (3) or 20 mM (4) glutamine. (C) Kinetic characterization of wild type and S48N S. typhimurium NAD synthetase. Initial rates were measured by spectrophotometrical coupled (SPEC) assays. The kinetic parameters Km and kcat are apparent values determined at fixed (saturating) concentrations of co-substrates. For fixed substrates, concentrations were: 2 mM ATP, 2 mM NaAD, and 40 mM NH3. Errors represent standard deviation.
Mentions: Salmonella nit mutants that were previously characterized as defective in nitrogen assimilation despite normal levels of ammonia assimilatory enzymes [36], provided us with a valuable case study to further address the question of the in vivo amide donor characteristic of NadE subfamily. Whereas both classes of mutants described in the original study, those induced by ICR (SK51) or by nitrosoguanidine (nit11), were genetically mapped within the nadE locus, the exact nature of these mutations has not been established. Amplification and sequencing of the respective mutant loci revealed two types of genetic lesions: (i) a deletion of a single nucleotide (G) in the promoter region upstream of the predicted -35 box, and (ii) a point mutation at the nucleotide 143 (G to A) replacing Ser-84 residue with Asn (Figure 4A). Therefore, a reported loss of the NADS activity could be expected to occur at the transcriptional level for the first type, and at the level of enzymatic properties for the second type of mutations. Indeed, a quantitative RT-PCR confirmed a substantial (∼ 100-fold) drop of the nadE mRNA level in SK51 compared to nit11 or wild-type S. typhimurium (Figure 4B). Importantly, increasing ammonia or glutamine in the media did not have any effect on the relative level of the nadE mRNA. It suggests that the observed suppression of growth phenotype is not due to the regulation on the level of transcription, but rather due to overcoming a competition for nitrogen source between a severely suppressed NADS and a robust glutamine synthetase (GlnA). The latter interpretation is consistent with the reported compensatory effect of the glnA mutation [36]. To determine the effect of the mutation nit11 on the enzyme function, wild type st_NADS and S48N mutant were overexpressed in E. coli, purified and their apparent steady-state kinetic parameters were determined toward ATP, NaAD, and NH3 (Figure 4C). The wild-type enzyme had kinetic parameters very close to those reported for E. coli NadE [52]. The most dramatic difference from S48N mutant was observed at the level of the apparent Km value for ammonia, which was ∼ 70-fold higher for the mutant st_NADS compared to the wild-type enzyme. This finding provides a straightforward interpretation for the observed growth phenotype of nit11 strain, which could be compensated for by increasing the concentration of ammonia in the media [36]. Notably, the mutated residue is a part of the conserved motif SGGXDST characteristic of the N-type ATP pyrophosphatase family [14]. Based on the NadE structure [34], it is located in the vicinity of the active site, which is consistent with the observed effect on the NADS activity.

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