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Characterization and improved properties of Glutamine synthetase from Providencia vermicola by site-directed mutagenesis
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...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199252/ https://www.ncbi.nlm.nih.gov/pubmed/30353099 http://dx.doi.org/10.1038/s41598-018-34022-5 |
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author | Zuo, Wu Nie, Leitong Baskaran, Ram Kumar, Ashok Liu, Ziduo |
author_facet | Zuo, Wu Nie, Leitong Baskaran, Ram Kumar, Ashok Liu, Ziduo |
author_sort | Zuo, Wu |
collection | PubMed |
description | 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-Na(2) 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-Na(2) 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. |
format | Online Article Text |
id | pubmed-6199252 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-61992522018-10-25 Characterization and improved properties of Glutamine synthetase from Providencia vermicola by site-directed mutagenesis Zuo, Wu Nie, Leitong Baskaran, Ram Kumar, Ashok Liu, Ziduo Sci Rep Article 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-Na(2) 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-Na(2) 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. Nature Publishing Group UK 2018-10-23 /pmc/articles/PMC6199252/ /pubmed/30353099 http://dx.doi.org/10.1038/s41598-018-34022-5 Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Zuo, Wu Nie, Leitong Baskaran, Ram Kumar, Ashok Liu, Ziduo Characterization and improved properties of Glutamine synthetase from Providencia vermicola by site-directed mutagenesis |
title | Characterization and improved properties of Glutamine synthetase from Providencia vermicola by site-directed mutagenesis |
title_full | Characterization and improved properties of Glutamine synthetase from Providencia vermicola by site-directed mutagenesis |
title_fullStr | Characterization and improved properties of Glutamine synthetase from Providencia vermicola by site-directed mutagenesis |
title_full_unstemmed | Characterization and improved properties of Glutamine synthetase from Providencia vermicola by site-directed mutagenesis |
title_short | Characterization and improved properties of Glutamine synthetase from Providencia vermicola by site-directed mutagenesis |
title_sort | characterization and improved properties of glutamine synthetase from providencia vermicola by site-directed mutagenesis |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199252/ https://www.ncbi.nlm.nih.gov/pubmed/30353099 http://dx.doi.org/10.1038/s41598-018-34022-5 |
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