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Effect of point mutations on Herbaspirillum seropedicae NifA activity.

Aquino B, Stefanello AA, Oliveira MA, Pedrosa FO, Souza EM, Monteiro RA, Chubatsu LS - Braz. J. Med. Biol. Res. (2015)

Bottom Line: Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins.However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium.This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.

ABSTRACT
NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

No MeSH data available.


Related in: MedlinePlus

Transcriptional activity of ΔN-NifA mutant proteins in Escherichiacoli JM109 (DE3) carrying pRT22 (nifH::lacZ). ΔN-NifAindicates NifA lacking 203 amino acid residues at the N-terminal GAF domain.ΔN-215D, ΔN-Q216I, and ΔN-S220I indicate the N-truncated forms of NifA mutants.β-galactosidase expression experiments were performed in NFDM medium supplementedwith 20 mM ammonium chloride (+N) or 0.2% casamino acids (-N) in the presence (+O)or absence (-O) of O2. Data are reported as the mean±SD of 3independent assays. β-galactosidase activity is reported as Miller units.
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f03: Transcriptional activity of ΔN-NifA mutant proteins in Escherichiacoli JM109 (DE3) carrying pRT22 (nifH::lacZ). ΔN-NifAindicates NifA lacking 203 amino acid residues at the N-terminal GAF domain.ΔN-215D, ΔN-Q216I, and ΔN-S220I indicate the N-truncated forms of NifA mutants.β-galactosidase expression experiments were performed in NFDM medium supplementedwith 20 mM ammonium chloride (+N) or 0.2% casamino acids (-N) in the presence (+O)or absence (-O) of O2. Data are reported as the mean±SD of 3independent assays. β-galactosidase activity is reported as Miller units.

Mentions: Considering that A215D, Q216I, and S220I are located in the central domain of NifA, andthat the full-length protein is inactive in E. coli (6) (Figure 2),these three mutations were also tested using an N-truncated form (GAF truncated protein)(Figure 3). The removal of the first 203 aminoacids of NifA yields an active protein in E. coli (8), as shown using protein expressed from plasmidpRAM2. The N-truncated protein (ΔN-NifA) was active regardless of the nitrogen level,but only under low O2, reinforcing its sensitivity toward O2. TheN-truncated ΔN-Q216I and ΔN-S220I mutants showed lower β-galactosidase activity thanthat expressed by pRAM2, indicating that these mutations negatively affecttranscriptional activity, while retaining O2 responsiveness. In contrast,ΔN-A215D was inactive under all tested conditions, suggesting that a negatively-chargedamino acid at position 215 affects the catalytic activity of the protein. These proteinswere expressed under all conditions tested, as determined by gel electrophoresis (datanot shown). The three disrupted amino acids are close to the ATP-binding site, which islocated at positions 231-238. In contrast to H. seropedicae, a NifAstrain with a mutation in this region (M217I) in S. meliloti was oxygentolerant (20).


Effect of point mutations on Herbaspirillum seropedicae NifA activity.

Aquino B, Stefanello AA, Oliveira MA, Pedrosa FO, Souza EM, Monteiro RA, Chubatsu LS - Braz. J. Med. Biol. Res. (2015)

Transcriptional activity of ΔN-NifA mutant proteins in Escherichiacoli JM109 (DE3) carrying pRT22 (nifH::lacZ). ΔN-NifAindicates NifA lacking 203 amino acid residues at the N-terminal GAF domain.ΔN-215D, ΔN-Q216I, and ΔN-S220I indicate the N-truncated forms of NifA mutants.β-galactosidase expression experiments were performed in NFDM medium supplementedwith 20 mM ammonium chloride (+N) or 0.2% casamino acids (-N) in the presence (+O)or absence (-O) of O2. Data are reported as the mean±SD of 3independent assays. β-galactosidase activity is reported as Miller units.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4541686&req=5

f03: Transcriptional activity of ΔN-NifA mutant proteins in Escherichiacoli JM109 (DE3) carrying pRT22 (nifH::lacZ). ΔN-NifAindicates NifA lacking 203 amino acid residues at the N-terminal GAF domain.ΔN-215D, ΔN-Q216I, and ΔN-S220I indicate the N-truncated forms of NifA mutants.β-galactosidase expression experiments were performed in NFDM medium supplementedwith 20 mM ammonium chloride (+N) or 0.2% casamino acids (-N) in the presence (+O)or absence (-O) of O2. Data are reported as the mean±SD of 3independent assays. β-galactosidase activity is reported as Miller units.
Mentions: Considering that A215D, Q216I, and S220I are located in the central domain of NifA, andthat the full-length protein is inactive in E. coli (6) (Figure 2),these three mutations were also tested using an N-truncated form (GAF truncated protein)(Figure 3). The removal of the first 203 aminoacids of NifA yields an active protein in E. coli (8), as shown using protein expressed from plasmidpRAM2. The N-truncated protein (ΔN-NifA) was active regardless of the nitrogen level,but only under low O2, reinforcing its sensitivity toward O2. TheN-truncated ΔN-Q216I and ΔN-S220I mutants showed lower β-galactosidase activity thanthat expressed by pRAM2, indicating that these mutations negatively affecttranscriptional activity, while retaining O2 responsiveness. In contrast,ΔN-A215D was inactive under all tested conditions, suggesting that a negatively-chargedamino acid at position 215 affects the catalytic activity of the protein. These proteinswere expressed under all conditions tested, as determined by gel electrophoresis (datanot shown). The three disrupted amino acids are close to the ATP-binding site, which islocated at positions 231-238. In contrast to H. seropedicae, a NifAstrain with a mutation in this region (M217I) in S. meliloti was oxygentolerant (20).

Bottom Line: Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins.However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium.This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.

ABSTRACT
NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

No MeSH data available.


Related in: MedlinePlus