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Binding and Catalytic Mechanisms of Veratryl Alcohol Oxidation by Lignin Peroxidase: A Theoretical and Experimental Study
Lignin peroxidase (LiP) and its natural substrate veratryl alcohol (VA) play a crucial role in lignin degradation by white-rot fungi. Understanding the molecular determinants for the interaction of this enzyme with its substrates is essential in the rational design of engineered peroxidases for biot...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Research Network of Computational and Structural Biotechnology
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6700493/ https://www.ncbi.nlm.nih.gov/pubmed/31452859 http://dx.doi.org/10.1016/j.csbj.2019.07.002 |
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author | Romero, Jefferson O. Fernández-Fueyo, Elena Avila-Salas, Fabián Recabarren, Rodrigo Alzate-Morales, Jans Martínez, Angel T. |
author_facet | Romero, Jefferson O. Fernández-Fueyo, Elena Avila-Salas, Fabián Recabarren, Rodrigo Alzate-Morales, Jans Martínez, Angel T. |
author_sort | Romero, Jefferson O. |
collection | PubMed |
description | Lignin peroxidase (LiP) and its natural substrate veratryl alcohol (VA) play a crucial role in lignin degradation by white-rot fungi. Understanding the molecular determinants for the interaction of this enzyme with its substrates is essential in the rational design of engineered peroxidases for biotechnological application. Here, we combine computational and experimental approaches to analyze the interaction of Phanerochaete chrysosporium LiP (isoenzyme H8) with VA and its radical cation (VA(•+), resulting from substrate oxidation by the enzyme). Interaction energy calculations at semiempirical quantum mechanical level (SQM) between LiP and VA/VA(•+) enabled to identify those residues at the acidic environment of catalytic Trp171 involved in the main interactions. Then, a battery of variants, with single and multiple mutations at these residues (Glu168, Asp165, Glu250, Asp264, and Phe267), was generated by directed mutagenesis, and their kinetics parameters were estimated on VA and two additional substrates. The experimental results show that Glu168 and Glu250 are crucial for the binding of VA, with Glu250 also contributing to the turnover of the enzyme. The experimental results were further rationalized through new calculations of interaction energies between VA/VA(•+) and LiP with each of the single mutations. Finally, the delocalization of spin density was determined with quantum mechanics/molecular mechanics calculations (QM/MM), further supporting the contribution of Glu250 to VA oxidation at Trp171. |
format | Online Article Text |
id | pubmed-6700493 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Research Network of Computational and Structural Biotechnology |
record_format | MEDLINE/PubMed |
spelling | pubmed-67004932019-08-26 Binding and Catalytic Mechanisms of Veratryl Alcohol Oxidation by Lignin Peroxidase: A Theoretical and Experimental Study Romero, Jefferson O. Fernández-Fueyo, Elena Avila-Salas, Fabián Recabarren, Rodrigo Alzate-Morales, Jans Martínez, Angel T. Comput Struct Biotechnol J Research Article Lignin peroxidase (LiP) and its natural substrate veratryl alcohol (VA) play a crucial role in lignin degradation by white-rot fungi. Understanding the molecular determinants for the interaction of this enzyme with its substrates is essential in the rational design of engineered peroxidases for biotechnological application. Here, we combine computational and experimental approaches to analyze the interaction of Phanerochaete chrysosporium LiP (isoenzyme H8) with VA and its radical cation (VA(•+), resulting from substrate oxidation by the enzyme). Interaction energy calculations at semiempirical quantum mechanical level (SQM) between LiP and VA/VA(•+) enabled to identify those residues at the acidic environment of catalytic Trp171 involved in the main interactions. Then, a battery of variants, with single and multiple mutations at these residues (Glu168, Asp165, Glu250, Asp264, and Phe267), was generated by directed mutagenesis, and their kinetics parameters were estimated on VA and two additional substrates. The experimental results show that Glu168 and Glu250 are crucial for the binding of VA, with Glu250 also contributing to the turnover of the enzyme. The experimental results were further rationalized through new calculations of interaction energies between VA/VA(•+) and LiP with each of the single mutations. Finally, the delocalization of spin density was determined with quantum mechanics/molecular mechanics calculations (QM/MM), further supporting the contribution of Glu250 to VA oxidation at Trp171. Research Network of Computational and Structural Biotechnology 2019-07-10 /pmc/articles/PMC6700493/ /pubmed/31452859 http://dx.doi.org/10.1016/j.csbj.2019.07.002 Text en © 2019 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Research Article Romero, Jefferson O. Fernández-Fueyo, Elena Avila-Salas, Fabián Recabarren, Rodrigo Alzate-Morales, Jans Martínez, Angel T. Binding and Catalytic Mechanisms of Veratryl Alcohol Oxidation by Lignin Peroxidase: A Theoretical and Experimental Study |
title | Binding and Catalytic Mechanisms of Veratryl Alcohol Oxidation by Lignin Peroxidase: A Theoretical and Experimental Study |
title_full | Binding and Catalytic Mechanisms of Veratryl Alcohol Oxidation by Lignin Peroxidase: A Theoretical and Experimental Study |
title_fullStr | Binding and Catalytic Mechanisms of Veratryl Alcohol Oxidation by Lignin Peroxidase: A Theoretical and Experimental Study |
title_full_unstemmed | Binding and Catalytic Mechanisms of Veratryl Alcohol Oxidation by Lignin Peroxidase: A Theoretical and Experimental Study |
title_short | Binding and Catalytic Mechanisms of Veratryl Alcohol Oxidation by Lignin Peroxidase: A Theoretical and Experimental Study |
title_sort | binding and catalytic mechanisms of veratryl alcohol oxidation by lignin peroxidase: a theoretical and experimental study |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6700493/ https://www.ncbi.nlm.nih.gov/pubmed/31452859 http://dx.doi.org/10.1016/j.csbj.2019.07.002 |
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