Characterization and improved properties of Glutamine synthetase from Providencia vermicola by site-directed mutagenesis
Abstract In this study, a novel gene for Glutamine synthetase was cloned and characterized for its activities and stabilities from a marine bacterium Providencia vermicola (PveGS). A mutant S54A was generated by site directed mutagenesis, which showed significant increase in the activity and stabili...
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| Autores principales: | , , , , |
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| Formato: | article |
| Lenguaje: | EN |
| Publicado: |
Nature Portfolio
2018
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| Materias: | |
| Acceso en línea: | https://doaj.org/article/f77a2f29af81430c8565fb0bab91aa75 |
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| Sumario: | Abstract In this study, a novel gene for Glutamine synthetase was cloned and characterized for its activities and stabilities from a marine bacterium Providencia vermicola (PveGS). A mutant S54A was generated by site directed mutagenesis, which showed significant increase in the activity and stabilities at a wide range of temperatures. The K m values of PveGS against hydroxylamine, ADP-Na2 and L-Glutamine were 15.7 ± 1.1, (25.2 ± 1.5) × 10−5 and 32.6 ± 1.7 mM, and the k cat were 17.0 ± 0.6, 9.14 ± 0.12 and 30.5 ± 1.0 s−1 respectively. In-silico-analysis revealed that the replacement of Ser at 54th position with Ala increased the catalytic activity of PveGS. Therefore, catalytic efficiency of mutant S54A had increased by 3.1, 0.89 and 2.9-folds towards hydroxylamine, ADP-Na2 and L-Glutamine respectively as compared to wild type. The structure prediction data indicated that the negatively charged pocket becomes enlarged and hydrogen bonding in Ser54 steadily promotes the product release. Interestingly, the residual activity of S54A mutant was increased by 10.7, 3.8 and 3.8 folds at 0, 10 and 50 °C as compared to WT. Structural analysis showed that S54A located on the loop near to the active site improved its flexibility due to the breaking of hydrogen bonds between product and enzyme. This also facilitated the enzyme to increase its cold adaptability as indicated by higher residual activity shown at 0 °C. Thus, replacement of Ala to Ser54 played a pivotal role to enhance the activities and stabilities at a wide range of temperatures. |
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