Tuning enzymatic activity by combining Virtual Docking and Residue Interaction Networks
Within the evolvability landscape of protein engineering approaches, the vast majority of mutations yield neutral, deleterious, or destabilizing effects. It has been demonstrated that stabilizing mutations are usually achieved by random mutagenesis, making the identification of improved protein vari...
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ftinrsquebec:oai:espace.inrs.ca:6715 2023-05-15T14:01:59+02:00 Tuning enzymatic activity by combining Virtual Docking and Residue Interaction Networks de los Santos, Yossef Lopez Fajardo, Ying Lian Chew Brault, Guillaume Doucet, Nicolas 2017 https://espace.inrs.ca/id/eprint/6715/ https://onlinelibrary.wiley.com/toc/1469896x/2017/26/S1 unknown de los Santos, Yossef Lopez, Fajardo, Ying Lian Chew, Brault, Guillaume et Doucet, Nicolas orcid:0000-0002-1952-9380 (2017). Tuning enzymatic activity by combining Virtual Docking and Residue Interaction Networks In: 31st Annual Symposium of the Protein-Society, July 24-27, 2017 Montreal, CANADA, Montréal (Québec) Canada. - Document issu d'une conférence ou d'un atelier Non évalué par les pairs 2017 ftinrsquebec 2023-02-10T11:44:11Z Within the evolvability landscape of protein engineering approaches, the vast majority of mutations yield neutral, deleterious, or destabilizing effects. It has been demonstrated that stabilizing mutations are usually achieved by random mutagenesis, making the identification of improved protein variants an exhaustive and inefficient process. Herein we present a semi-rational combinatorial approach supported by docking simulations and Residue Interaction Networks (RINs) to design smaller and smarter libraries of mutants. Lipase B from Pseudozyma antarctica (CalB) was selected as an industrially relevant model system. Since CalB displays very low activity towards bulky substrates, the main goal of this project was to develop CalB variants with enhanced synthetic activity towards aromatic substrates like cinnamic and salicylic acid. We used this combined approach to uncover the importance of residues in the CalB active-site cavity and their contribution to the synthetic reaction (Docking), in addition to calculating the energetic contributions upon site-directed mutagenesis (RINs). As a result, we improved the synthetic activity of CalB from 2% to more than 70% of the total substrate-product conversion ratio. This strategy allowed us to obtain more than 5 CalB variants with enhanced activity toward two bulky substrates after only two rounds of directed evolution. The sequential incorporation of favorable mutations increased our chances of selecting improved CalB variants and reduced screening effort. The use of a ‘bottom-up’ strategy such as the RINs allowed us to further understand the effects of mutations throughout the protein structure, a powerful tool for protein engineering purposes. Other/Unknown Material Antarc* Antarctica Institut national de la recherche scientifique, Québec: Espace INRS |
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Institut national de la recherche scientifique, Québec: Espace INRS |
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- de los Santos, Yossef Lopez Fajardo, Ying Lian Chew Brault, Guillaume Doucet, Nicolas Tuning enzymatic activity by combining Virtual Docking and Residue Interaction Networks |
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description |
Within the evolvability landscape of protein engineering approaches, the vast majority of mutations yield neutral, deleterious, or destabilizing effects. It has been demonstrated that stabilizing mutations are usually achieved by random mutagenesis, making the identification of improved protein variants an exhaustive and inefficient process. Herein we present a semi-rational combinatorial approach supported by docking simulations and Residue Interaction Networks (RINs) to design smaller and smarter libraries of mutants. Lipase B from Pseudozyma antarctica (CalB) was selected as an industrially relevant model system. Since CalB displays very low activity towards bulky substrates, the main goal of this project was to develop CalB variants with enhanced synthetic activity towards aromatic substrates like cinnamic and salicylic acid. We used this combined approach to uncover the importance of residues in the CalB active-site cavity and their contribution to the synthetic reaction (Docking), in addition to calculating the energetic contributions upon site-directed mutagenesis (RINs). As a result, we improved the synthetic activity of CalB from 2% to more than 70% of the total substrate-product conversion ratio. This strategy allowed us to obtain more than 5 CalB variants with enhanced activity toward two bulky substrates after only two rounds of directed evolution. The sequential incorporation of favorable mutations increased our chances of selecting improved CalB variants and reduced screening effort. The use of a ‘bottom-up’ strategy such as the RINs allowed us to further understand the effects of mutations throughout the protein structure, a powerful tool for protein engineering purposes. |
format |
Other/Unknown Material |
author |
de los Santos, Yossef Lopez Fajardo, Ying Lian Chew Brault, Guillaume Doucet, Nicolas |
author_facet |
de los Santos, Yossef Lopez Fajardo, Ying Lian Chew Brault, Guillaume Doucet, Nicolas |
author_sort |
de los Santos, Yossef Lopez |
title |
Tuning enzymatic activity by combining Virtual Docking and Residue Interaction Networks |
title_short |
Tuning enzymatic activity by combining Virtual Docking and Residue Interaction Networks |
title_full |
Tuning enzymatic activity by combining Virtual Docking and Residue Interaction Networks |
title_fullStr |
Tuning enzymatic activity by combining Virtual Docking and Residue Interaction Networks |
title_full_unstemmed |
Tuning enzymatic activity by combining Virtual Docking and Residue Interaction Networks |
title_sort |
tuning enzymatic activity by combining virtual docking and residue interaction networks |
publishDate |
2017 |
url |
https://espace.inrs.ca/id/eprint/6715/ https://onlinelibrary.wiley.com/toc/1469896x/2017/26/S1 |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_relation |
de los Santos, Yossef Lopez, Fajardo, Ying Lian Chew, Brault, Guillaume et Doucet, Nicolas orcid:0000-0002-1952-9380 (2017). Tuning enzymatic activity by combining Virtual Docking and Residue Interaction Networks In: 31st Annual Symposium of the Protein-Society, July 24-27, 2017 Montreal, CANADA, Montréal (Québec) Canada. |
_version_ |
1766272044293423104 |