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:	Wu Zuo, Leitong Nie, Ram Baskaran, Ashok Kumar, Ziduo Liu
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2018
Materias:	Vermicola Glutamine Synthetase Structure Prediction Data Cold Adaptability Maximum Allowable Area Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f77a2f29af81430c8565fb0bab91aa75
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spelling	oai:doaj.org-article:f77a2f29af81430c8565fb0bab91aa752021-12-02T15:07:52ZCharacterization and improved properties of Glutamine synthetase from Providencia vermicola by site-directed mutagenesis10.1038/s41598-018-34022-52045-2322https://doaj.org/article/f77a2f29af81430c8565fb0bab91aa752018-10-01T00:00:00Zhttps://doi.org/10.1038/s41598-018-34022-5https://doaj.org/toc/2045-2322Abstract 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.Wu ZuoLeitong NieRam BaskaranAshok KumarZiduo LiuNature PortfolioarticleVermicolaGlutamine SynthetaseStructure Prediction DataCold AdaptabilityMaximum Allowable AreaMedicineRScienceQENScientific Reports, Vol 8, Iss 1, Pp 1-8 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	Vermicola Glutamine Synthetase Structure Prediction Data Cold Adaptability Maximum Allowable Area Medicine R Science Q
spellingShingle	Vermicola Glutamine Synthetase Structure Prediction Data Cold Adaptability Maximum Allowable Area Medicine R Science Q Wu Zuo Leitong Nie Ram Baskaran Ashok Kumar Ziduo Liu Characterization and improved properties of Glutamine synthetase from Providencia vermicola by site-directed mutagenesis
description	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.
format	article
author	Wu Zuo Leitong Nie Ram Baskaran Ashok Kumar Ziduo Liu
author_facet	Wu Zuo Leitong Nie Ram Baskaran Ashok Kumar Ziduo Liu
author_sort	Wu Zuo
title	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_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_sort	characterization and improved properties of glutamine synthetase from providencia vermicola by site-directed mutagenesis
publisher	Nature Portfolio
publishDate	2018
url	https://doaj.org/article/f77a2f29af81430c8565fb0bab91aa75
work_keys_str_mv	AT wuzuo characterizationandimprovedpropertiesofglutaminesynthetasefromprovidenciavermicolabysitedirectedmutagenesis AT leitongnie characterizationandimprovedpropertiesofglutaminesynthetasefromprovidenciavermicolabysitedirectedmutagenesis AT rambaskaran characterizationandimprovedpropertiesofglutaminesynthetasefromprovidenciavermicolabysitedirectedmutagenesis AT ashokkumar characterizationandimprovedpropertiesofglutaminesynthetasefromprovidenciavermicolabysitedirectedmutagenesis AT ziduoliu characterizationandimprovedpropertiesofglutaminesynthetasefromprovidenciavermicolabysitedirectedmutagenesis
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Characterization and improved properties of Glutamine synthetase from Providencia vermicola by site-directed mutagenesis

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