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Characterization of an L-phosphinothricin resistant glutamine synthetase from Exiguobacterium sp. and its improvement

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Abstract

A glutamine synthetase (GS; 1341 bp) gene with potent L-phosphinothricin (PPT) resistance was isolated and characterized from a marine bacterium Exiguobacterium sp. Molecular docking analysis indicated that the substitution of residues Glu60 and Arg64 may lead to significant changes in binding pocket. To enhance the enzymatic property of GS, variants E60A and R64G were obtained by site-directed mutagenesis. The results revealed a noteworthy change in the thermostability and activity in comparison to the wild type (WT). WT exhibited optimum activity at 35 °C, while E60A and R64G exhibited optimum activity at 45 and 40 °C, respectively. The mutant R64G was 4.3 times more stable at 70 °C in comparison to WT, while E60A was 5.7 times more stable. Kinetic analysis revealed that the k cat value of R64G mutant was 8.10-, 7.25- and 7.63-fold that of WT for ADP, glutamine and hydroxylamine, respectively. The kinetic inhibition (K i, 4.91 ± 0.42 mM) of R64G was 2.02-fold that of WT (2.43 ± 0.14 mM) for L-phosphinothricin. The analysis of structure and function relationship showed that the binding pocket underwent dramatic changes when Arg site of 64 was substituted by Gly, thus promoting the rapid capture of substrates and leading to increase in activity and PPT-resistance of mutant R64G. The rearrangements of the residues at the molecular level formed new hydrogen bonds around the active site, which contributed to the increase of thermostability of enzymes. This study provides new insights into substrate binding mechanism of glutamine synthetase and the improved GS gene also has a potential for application in transgenic crops with L-phosphinothricin tolerance.

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Acknowledgements

This work was supported by grants from the Genetically Modified Organisms Breeding Major Projects of China (2016ZX08001001001-001-001-004).

Author contribution statement

SWZ designed and performed the experiments and drafted this manuscript. YKH, AK and HG contributed to characterization, expression experiments and revision of the manuscript. NH and ZDL are corresponding authors, who conceived, designed and supervised the experiments. All authors have read and approved the manuscript.

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Authors and Affiliations

  1. College of Life Science and Technology, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, 430070, China

    Shaowei Zhang, Yingkun Han, Ashok Kumar & Ziduo Liu

  2. College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211800, People’s Republic of China

    Haofeng Gao & Nan Hu

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  1. Shaowei Zhang
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  2. Yingkun Han
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  3. Ashok Kumar
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  4. Haofeng Gao
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  5. Ziduo Liu
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  6. Nan Hu
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Correspondence to Ziduo Liu or Nan Hu.

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Zhang, S., Han, Y., Kumar, A. et al. Characterization of an L-phosphinothricin resistant glutamine synthetase from Exiguobacterium sp. and its improvement. Appl Microbiol Biotechnol 101, 3653–3661 (2017). https://doi.org/10.1007/s00253-017-8103-1

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