Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense.

Fernanda Ghenov,Roseli Wassem,Luciano Fernandes Huergo,Edileusa Cristina Marques Gerhardt,Emanuel Maltempi Souza,Fabio Oliveira Pedrosa

doi:10.1590/1519-6984.235927

Abstract

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.

Highlights

In plants, nitrogen starvation is associated with reduction of cell division and expansion, leaf area and photosynthesis (Lawlor David, 2002)
Due to its higher solubility, much larger amounts of purified wild type glutamine synthetase (GS) were obtained than was the case for position 347 for a leucine (P347L)-GS
Visual comparisons of SDS-PAGE gels of protein extracts made from cultures induced for 18 h at 16 oC, suggest that wild-type GS was ≈ 50% soluble, whereas the mutant was almost completely insoluble

Summary

Introduction

Nitrogen starvation is associated with reduction of cell division and expansion, leaf area and photosynthesis (Lawlor David, 2002). Plants can use as nitrogen sources ammonium, nitrate and amino acids, but cannot incorporate the most abundant form of nitrogen available on earth, dinitrogen (N2). Biological fixation of nitrogen is the reduction of dinitrogen gas into ammonium by the nitrogenase complex present in a restricted group of prokaryotes. Biological nitrogen fixation is negatively controlled by the availability of ammonium (Hartmann et al, 1986; Merrick and Edwards, 1995). The glutamate synthase enzyme (GOGAT), encoded by the gltD and gltB genes, catalyse the reductive transfer of the amide group from L-glutamine to α-ketoglutarate, producing two L-glutamate molecules in an NADPH-dependent reaction (Merrick and Edwards, 1995; Westby et al, 1987)

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Conclusion