Characterization And Improvement Of Glutamine Synthetase From Exiguobacterium Sp. N10-1

Glutamine synthetase(GS; EC 6.3.1.2) is an important enzyme which catalyzes the conversion of glutamate and ammonia into L-glutamine in an energy dependent reaction with the simultaneous hydrolysis of ATP to ADP. GS can be used to synthesis L-glutamine and L-theanine in industry. Besides, overexpression of GS can increase the tolerance towards L-phosphinothricin in transgenic plants. It also has potential to improve qualities/the properties of crop plants, such as nitrogen-deficiency tolerance and high-salt-stress tolerance.In this study, Exiguobacterium sp. N10-1 was screened from bacterial in special habitats. A glutamine synthetase(GS; 1341 bp) gene with potent L-phosphinothricin(PPT) resistance was isolated and characterized from Exiguobacterium sp. N10-1. The purified ExiGS(WT) encoded a polypeptide chain of 446 amino acids with calculated molecular mass of 50.1 kDa on SDS-PAGE. Molecular docking analyses indicated that the substitution of residues Glu60 and Arg64 may result significant changes of binding pocket. To improve the enzymatic property of GS, variants E60 A and R64 G were obtained by site-directed mutagenesis. The effect of temperature on the activity of purified GS proteins(WT and mutants) was determined and WT showed the optimum activity at 35℃ versus 45, 40 and 50℃ for E60 A, R64 G and E60A/R64 G, respectively. The thermal stability of WT and mutants E60 A, R64 G and E60A/R64 G was studied at 50, 60 and 70℃, and the R64 G variant was dramatically thermostable at all tested temperatures as compared to WT and the other variants. The WT showed 50% decrease in its original activity after 1 and 0.5 h at 60 and 70℃, respectively. All GS proteins(WT and mutants) retained their original activities at 50℃ for 3 h. However, a rapid decrease in the activity of WT was recorded at 60℃ after 3 h incubation. At 70℃, the E60 A and R64 G mutants retained approximately 40% of their original activities after 3 h, but a rapid decrease occurred in the activities of WT and E60A/R64 G.Kinetic analysis showed that the kcat value of R64 G mutant was 8.10-, 7.25- and 7.63-fold that of WT for ADP, glutamine and hydroxylamine, respectively. The kinetic inhibition(Ki, 4.91± 0.42 mM) of R64 G was 2.02-fold that of WT(2.43± 0.14 mM) for L-phosphinothricin.The analysis of structure and function relationship revealed that the binding pocket underwent dramatically changes when Arg site of 64 was substituted by Gly, thus promoting the rapid capture of substrates and leading the increase of activity and PPT-resistance of mutant R64 G. 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 of substrate binding mechanism of glutamine synthetase and the improved GS gene also has a potential for application in transgenic crops with L-phosphinothricin tolerance.