Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of <i>Azospirillum brasilense</i>

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

Keywords:
nitrogen fixation; adenylylation; adenilyltransferase; EC 6.3.1.2; GlnE

Authorship

Fernanda Ghenov

Universidade Federal do Paraná – UFPR, Departamento de Bioquímica e Biologia Molecular, Núcleo de Fixação Biológica de Nitrogênio, Curitiba, PR, BrasilUniversidade Federal do ParanáBrasilCuritiba, PR, BrasilUniversidade Federal do Paraná – UFPR, Departamento de Bioquímica e Biologia Molecular, Núcleo de Fixação Biológica de Nitrogênio, Curitiba, PR, Brasil

http://orcid.org/0000-0001-8250-4015

Edileusa Cristina Marques Gerhardt

http://orcid.org/0000-0001-7088-1681

Luciano Fernandes Huergo

Universidade Federal do Paraná – UFPR, Setor Litoral, Caiobá Matinhos, PR, BrasilUniversidade Federal do ParanáBrasilCaiobá Matinhos, PR, BrasilUniversidade Federal do Paraná – UFPR, Setor Litoral, Caiobá Matinhos, PR, Brasil

http://orcid.org/0000-0002-7587-9510

Fabio Oliveira Pedrosa

http://orcid.org/0000-0003-3711-969X

Roseli Wassem

Universidade Federal do Paraná – UFPR, Departamento de Genética, Curitiba, PR, BrasilUniversidade Federal do ParanáBrasilCuritiba, PR, BrasilUniversidade Federal do Paraná – UFPR, Departamento de Genética, Curitiba, PR, Brasil

http://orcid.org/0000-0002-2469-4479

Emanuel Maltempi Souza ^**e-mail: souzaem@ufpr.br

http://orcid.org/0000-0003-1546-9218

*e-mail: souzaem@ufpr.br

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (3)
Tables (1)

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Figure 1
Transferase activity of glutamine synthetase. (A) Transferase activity of wild-type glutamine synthetase in the absence and presence of magnesium as well as with snake venom phosphodiesterase treatment; (B) Transferase activity of P347L glutamine synthetase in the absence and presence of magnesium, and with snake venom phosphodiesterase treatment. The activity of GS is expressed in μmol γ-glutamyl-hydroxamate.min^-1.mg protein^-1, given that the absorbance of 530 nm of 1 µmol γ-glutamyl-hydroxamate was 0.054. The total activity was determined in the absence of Mg²⁺ (-Mg²⁺) and the non-adenylylated (active) fraction was determined in the presence of 60 mM Mg²⁺ (+Mg²⁺). Samples were incubated at 30 ºC for 0, 10, 30 and 60 min before measuring activity. SVP-treated GS samples (+ SVP) were incubated with snake venom phosphodiesterase. GS activity reactions contained 3 µg of protein.

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Figure 2
Western blot assays of glutamine synthetase after treatment with snake venom phosphodiesterase. Samples (~ 0.3 µg GS protein) were separated by SDS‐PAGE followed by Western blotting with an anti‐GS antibody. A) Wild-type glutamine synthetase; B) P347L glutamine synthetase. Lane 1: GS after 0 min of incubation at 30 ºC without any treatment; lanes 2 to 5: GS after 0, 10, 30 and 60 min incubation at 30 ºC with snake venom phosphodiesterase. Lane 6: GS after 60 min incubation at 30 ºC without treatment.

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Figure 3
Prediction of the structure of glutamine synthetase from the mutant P347L. (A) Prediction of the P347L-GS structure. The amino acid marked in pink corresponds to leucine in strain HM053; (B) b1) Prediction structure of wild-type GS from amino acid 346 to 361. b2) Prediction structure of P347L-GS from amino acid 346 to 361. b3) Alignment of prediction structures of wildtype GS and P347L GS from amino acid 346 to 361. The amino acid marked in blue corresponds to the proline that is mutated in strain HM053. The amino acid marked in pink is leucine that replaced proline in the mutated amino acid in strain HM053.

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Table 1
Proteins identified by MALDI-TOF mass spectroscopy.

Figure 1 Transferase activity of glutamine synthetase. (A) Transferase activity of wild-type glutamine synthetase in the absence and presence of magnesium as well as with snake venom phosphodiesterase treatment; (B) Transferase activity of P347L glutamine synthetase in the absence and presence of magnesium, and with snake venom phosphodiesterase treatment. The activity of GS is expressed in μmol γ-glutamyl-hydroxamate.min^-1.mg protein^-1, given that the absorbance of 530 nm of 1 µmol γ-glutamyl-hydroxamate was 0.054. The total activity was determined in the absence of Mg²⁺ (-Mg²⁺) and the non-adenylylated (active) fraction was determined in the presence of 60 mM Mg²⁺ (+Mg²⁺). Samples were incubated at 30 ºC for 0, 10, 30 and 60 min before measuring activity. SVP-treated GS samples (+ SVP) were incubated with snake venom phosphodiesterase. GS activity reactions contained 3 µg of protein.

Figure 2 Western blot assays of glutamine synthetase after treatment with snake venom phosphodiesterase. Samples (~ 0.3 µg GS protein) were separated by SDS‐PAGE followed by Western blotting with an anti‐GS antibody. A) Wild-type glutamine synthetase; B) P347L glutamine synthetase. Lane 1: GS after 0 min of incubation at 30 ºC without any treatment; lanes 2 to 5: GS after 0, 10, 30 and 60 min incubation at 30 ºC with snake venom phosphodiesterase. Lane 6: GS after 60 min incubation at 30 ºC without treatment.

Figure 3 Prediction of the structure of glutamine synthetase from the mutant P347L. (A) Prediction of the P347L-GS structure. The amino acid marked in pink corresponds to leucine in strain HM053; (B) b1) Prediction structure of wild-type GS from amino acid 346 to 361. b2) Prediction structure of P347L-GS from amino acid 346 to 361. b3) Alignment of prediction structures of wildtype GS and P347L GS from amino acid 346 to 361. The amino acid marked in blue corresponds to the proline that is mutated in strain HM053. The amino acid marked in pink is leucine that replaced proline in the mutated amino acid in strain HM053.

Table 1 Proteins identified by MALDI-TOF mass spectroscopy.

Sample	Protein identified	Mascot MOWSE Score	M.W. (kDa)*	# Peptides identified	Coverage
Wild-type GS	Glutamine synthetase	131	52.3	12	38%
P347L-GS	Glutamine synthetase	141	52.3	13	33%

^*
Molecular weight.

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Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of <i>Azospirillum brasilense</i>

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[1] *e-mail: souzaem@ufpr.br