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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

Scheme of Herbaspirillum seropedicae NifA domains. Numbers indicate the aminoacid position on the primary structure. The N-terminal GAF domain, central domain,and C-terminal domain are indicated as open rectangles. The ATP-binding motifs,located in the central domain, and the helix-turn-helix (HTH) motif in theC-terminal domain are indicated as gray rectangles, and were predicted using theScanProsite tool (http://prosite.expasy.org/scanprosite) (29) and Gym2.0 (30),respectively. Point-mutations (K22V, G25E, T160E, M161V, L172R, A215D, Q216I, andS220I) are indicated by gray triangles in the N-terminal GAF and central domainsof NifA.
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f01: Scheme of Herbaspirillum seropedicae NifA domains. Numbers indicate the aminoacid position on the primary structure. The N-terminal GAF domain, central domain,and C-terminal domain are indicated as open rectangles. The ATP-binding motifs,located in the central domain, and the helix-turn-helix (HTH) motif in theC-terminal domain are indicated as gray rectangles, and were predicted using theScanProsite tool (http://prosite.expasy.org/scanprosite) (29) and Gym2.0 (30),respectively. Point-mutations (K22V, G25E, T160E, M161V, L172R, A215D, Q216I, andS220I) are indicated by gray triangles in the N-terminal GAF and central domainsof NifA.

Mentions: The H. seropedicae NifA N-terminal GAF domain comprises the first 184amino acids (Figure 1), and although it isinvolved in negative regulation by ammonium, it is not strictly required for activationof nif gene transcription (6,8). This N-terminal GAF domaininteracts with GlnK in response to the fixed nitrogen concentration (9). The N-terminal GAF domain is linked to thecentral domain by the 18 amino acids of the Q-linker. The H.seropedicae NifA central domain comprises 236 amino acids and contains thecatalytic site. It also interacts with the σ54 RNA polymerase holoenzyme(4). The central domain is linked to theC-terminal domain by a 58-amino acid region named the ID-linker. A conserved cysteinemotif located at the end of the central domain and the ID-linker (positions 414, 426,446, and 451) is suggested to be involved in the regulation of NifA by O2.Mutation of these cysteine residues produces inactive proteins (10). Finally, the last 43 amino acids of the NifA primary sequenceform the C-terminal domain, which is responsible for DNA binding (11).


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)

Scheme of Herbaspirillum seropedicae NifA domains. Numbers indicate the aminoacid position on the primary structure. The N-terminal GAF domain, central domain,and C-terminal domain are indicated as open rectangles. The ATP-binding motifs,located in the central domain, and the helix-turn-helix (HTH) motif in theC-terminal domain are indicated as gray rectangles, and were predicted using theScanProsite tool (http://prosite.expasy.org/scanprosite) (29) and Gym2.0 (30),respectively. Point-mutations (K22V, G25E, T160E, M161V, L172R, A215D, Q216I, andS220I) are indicated by gray triangles in the N-terminal GAF and central domainsof NifA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f01: Scheme of Herbaspirillum seropedicae NifA domains. Numbers indicate the aminoacid position on the primary structure. The N-terminal GAF domain, central domain,and C-terminal domain are indicated as open rectangles. The ATP-binding motifs,located in the central domain, and the helix-turn-helix (HTH) motif in theC-terminal domain are indicated as gray rectangles, and were predicted using theScanProsite tool (http://prosite.expasy.org/scanprosite) (29) and Gym2.0 (30),respectively. Point-mutations (K22V, G25E, T160E, M161V, L172R, A215D, Q216I, andS220I) are indicated by gray triangles in the N-terminal GAF and central domainsof NifA.
Mentions: The H. seropedicae NifA N-terminal GAF domain comprises the first 184amino acids (Figure 1), and although it isinvolved in negative regulation by ammonium, it is not strictly required for activationof nif gene transcription (6,8). This N-terminal GAF domaininteracts with GlnK in response to the fixed nitrogen concentration (9). The N-terminal GAF domain is linked to thecentral domain by the 18 amino acids of the Q-linker. The H.seropedicae NifA central domain comprises 236 amino acids and contains thecatalytic site. It also interacts with the σ54 RNA polymerase holoenzyme(4). The central domain is linked to theC-terminal domain by a 58-amino acid region named the ID-linker. A conserved cysteinemotif located at the end of the central domain and the ID-linker (positions 414, 426,446, and 451) is suggested to be involved in the regulation of NifA by O2.Mutation of these cysteine residues produces inactive proteins (10). Finally, the last 43 amino acids of the NifA primary sequenceform the C-terminal domain, which is responsible for DNA binding (11).

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