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Two Structurally Different Dienelactone Hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 Plasmid pJP4 Catalyse Cis- and Trans-Dienelactones with Similar Efficiency

In this study, dienelactone hydrolases (TfdEI and TfdEII) located on plasmid pJP4 of Cupriavidus necator JMP134 were cloned, purified, characterized and three dimensional structures were predicted. tfdEI and tfdEII genes were cloned into pET21b vector and expressed in E. coli BL21(DE3). The enzymes...

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Autores principales: Kumar, Ajit, Pillay, Balakrishna, Olaniran, Ademola O.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4108320/
https://www.ncbi.nlm.nih.gov/pubmed/25054964
http://dx.doi.org/10.1371/journal.pone.0101801
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author Kumar, Ajit
Pillay, Balakrishna
Olaniran, Ademola O.
author_facet Kumar, Ajit
Pillay, Balakrishna
Olaniran, Ademola O.
author_sort Kumar, Ajit
collection PubMed
description In this study, dienelactone hydrolases (TfdEI and TfdEII) located on plasmid pJP4 of Cupriavidus necator JMP134 were cloned, purified, characterized and three dimensional structures were predicted. tfdEI and tfdEII genes were cloned into pET21b vector and expressed in E. coli BL21(DE3). The enzymes were purified by applying ultra-membrane filtration, anion-exchange QFF and gel-filtration columns. The enzyme activity was determined by using cis-dienelactone. The three-dimensional structure of enzymes was predicted using SWISS-MODEL workspace and the biophysical properties were determined on ExPASy server. Both TfdEI and TfdEII (M(r) 25 kDa) exhibited optimum activity at 37°C and pH 7.0. The enzymes retained approximately 50% of their activity after 1 h of incubation at 50°C and showed high stability against denaturing agents. The TfdEI and TfdEII hydrolysed cis-dienelactone at a rate of 0.258 and 0.182 µMs(−1), with a K(m) value of 87 µM and 305 µM, respectively. Also, TfdEI and TfdEII hydrolysed trans-dienelactone at a rate of 0.053 µMs(−1) and 0.0766 µMs(−1), with a K(m) value of 84 µM and 178 µM, respectively. The TfdEI and TfdEII k(cat)/K(m) ratios were 0.12 µM(−1)s(−1)and 0.13 µM(−1)s(−1) and 0.216 µM(−1)s(−1) and 0.094 µM(−1)s(−1) for for cis- and trans-dienelactone, respectively. The k(cat)/K(m) ratios for cis-dienelactone show that both enzymes catalyse the reaction with same efficiency even though K(m) value differs significantly. This is the first report to characterize and compare reaction kinetics of purified TfdEI and TfdEII from Cupriavidus necator JMP134 and may be helpful for further exploration of their catalytic mechanisms.
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spelling pubmed-41083202014-07-24 Two Structurally Different Dienelactone Hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 Plasmid pJP4 Catalyse Cis- and Trans-Dienelactones with Similar Efficiency Kumar, Ajit Pillay, Balakrishna Olaniran, Ademola O. PLoS One Research Article In this study, dienelactone hydrolases (TfdEI and TfdEII) located on plasmid pJP4 of Cupriavidus necator JMP134 were cloned, purified, characterized and three dimensional structures were predicted. tfdEI and tfdEII genes were cloned into pET21b vector and expressed in E. coli BL21(DE3). The enzymes were purified by applying ultra-membrane filtration, anion-exchange QFF and gel-filtration columns. The enzyme activity was determined by using cis-dienelactone. The three-dimensional structure of enzymes was predicted using SWISS-MODEL workspace and the biophysical properties were determined on ExPASy server. Both TfdEI and TfdEII (M(r) 25 kDa) exhibited optimum activity at 37°C and pH 7.0. The enzymes retained approximately 50% of their activity after 1 h of incubation at 50°C and showed high stability against denaturing agents. The TfdEI and TfdEII hydrolysed cis-dienelactone at a rate of 0.258 and 0.182 µMs(−1), with a K(m) value of 87 µM and 305 µM, respectively. Also, TfdEI and TfdEII hydrolysed trans-dienelactone at a rate of 0.053 µMs(−1) and 0.0766 µMs(−1), with a K(m) value of 84 µM and 178 µM, respectively. The TfdEI and TfdEII k(cat)/K(m) ratios were 0.12 µM(−1)s(−1)and 0.13 µM(−1)s(−1) and 0.216 µM(−1)s(−1) and 0.094 µM(−1)s(−1) for for cis- and trans-dienelactone, respectively. The k(cat)/K(m) ratios for cis-dienelactone show that both enzymes catalyse the reaction with same efficiency even though K(m) value differs significantly. This is the first report to characterize and compare reaction kinetics of purified TfdEI and TfdEII from Cupriavidus necator JMP134 and may be helpful for further exploration of their catalytic mechanisms. Public Library of Science 2014-07-23 /pmc/articles/PMC4108320/ /pubmed/25054964 http://dx.doi.org/10.1371/journal.pone.0101801 Text en © 2014 Kumar et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Kumar, Ajit
Pillay, Balakrishna
Olaniran, Ademola O.
Two Structurally Different Dienelactone Hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 Plasmid pJP4 Catalyse Cis- and Trans-Dienelactones with Similar Efficiency
title Two Structurally Different Dienelactone Hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 Plasmid pJP4 Catalyse Cis- and Trans-Dienelactones with Similar Efficiency
title_full Two Structurally Different Dienelactone Hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 Plasmid pJP4 Catalyse Cis- and Trans-Dienelactones with Similar Efficiency
title_fullStr Two Structurally Different Dienelactone Hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 Plasmid pJP4 Catalyse Cis- and Trans-Dienelactones with Similar Efficiency
title_full_unstemmed Two Structurally Different Dienelactone Hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 Plasmid pJP4 Catalyse Cis- and Trans-Dienelactones with Similar Efficiency
title_short Two Structurally Different Dienelactone Hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 Plasmid pJP4 Catalyse Cis- and Trans-Dienelactones with Similar Efficiency
title_sort two structurally different dienelactone hydrolases (tfdei and tfdeii) from cupriavidus necator jmp134 plasmid pjp4 catalyse cis- and trans-dienelactones with similar efficiency
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4108320/
https://www.ncbi.nlm.nih.gov/pubmed/25054964
http://dx.doi.org/10.1371/journal.pone.0101801
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