Cargando…

Mapping Potential Determinants of Peroxidative Activity in an Evolved Fungal Peroxygenase from Agrocybe aegerita

Fungal unspecific peroxygenases (UPOs) are hybrid biocatalysts with peroxygenative activity that insert oxygen into non-activated compounds, while also possessing convergent peroxidative activity for one electron oxidation reactions. In several ligninolytic peroxidases, the site of peroxidative acti...

Descripción completa

Detalles Bibliográficos
Autores principales: Molina-Espeja, Patricia, Beltran-Nogal, Alejandro, Alfuzzi, Maria Alejandra, Guallar, Victor, Alcalde, Miguel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8476742/
https://www.ncbi.nlm.nih.gov/pubmed/34595162
http://dx.doi.org/10.3389/fbioe.2021.741282
_version_ 1784575681717338112
author Molina-Espeja, Patricia
Beltran-Nogal, Alejandro
Alfuzzi, Maria Alejandra
Guallar, Victor
Alcalde, Miguel
author_facet Molina-Espeja, Patricia
Beltran-Nogal, Alejandro
Alfuzzi, Maria Alejandra
Guallar, Victor
Alcalde, Miguel
author_sort Molina-Espeja, Patricia
collection PubMed
description Fungal unspecific peroxygenases (UPOs) are hybrid biocatalysts with peroxygenative activity that insert oxygen into non-activated compounds, while also possessing convergent peroxidative activity for one electron oxidation reactions. In several ligninolytic peroxidases, the site of peroxidative activity is associated with an oxidizable aromatic residue at the protein surface that connects to the buried heme domain through a long-range electron transfer (LRET) pathway. However, the peroxidative activity of these enzymes may also be initiated at the heme access channel. In this study, we examined the origin of the peroxidative activity of UPOs using an evolved secretion variant (PaDa-I mutant) from Agrocybe aegerita as our point of departure. After analyzing potential radical-forming aromatic residues at the PaDa-I surface by QM/MM, independent saturation mutagenesis libraries of Trp24, Tyr47, Tyr79, Tyr151, Tyr265, Tyr281, Tyr293 and Tyr325 were constructed and screened with both peroxidative and peroxygenative substrates. These mutant libraries were mostly inactive, with only a few functional clones detected, none of these showing marked differences in the peroxygenative and peroxidative activities. By contrast, when the flexible Gly314-Gly318 loop that is found at the outer entrance to the heme channel was subjected to combinatorial saturation mutagenesis and computational analysis, mutants with improved kinetics and a shift in the pH activity profile for peroxidative substrates were found, while they retained their kinetic values for peroxygenative substrates. This striking change was accompanied by a 4.5°C enhancement in kinetic thermostability despite the variants carried up to four consecutive mutations. Taken together, our study proves that the origin of the peroxidative activity in UPOs, unlike other ligninolytic peroxidases described to date, is not dependent on a LRET route from oxidizable residues at the protein surface, but rather it seems to be exclusively located at the heme access channel.
format Online
Article
Text
id pubmed-8476742
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-84767422021-09-29 Mapping Potential Determinants of Peroxidative Activity in an Evolved Fungal Peroxygenase from Agrocybe aegerita Molina-Espeja, Patricia Beltran-Nogal, Alejandro Alfuzzi, Maria Alejandra Guallar, Victor Alcalde, Miguel Front Bioeng Biotechnol Bioengineering and Biotechnology Fungal unspecific peroxygenases (UPOs) are hybrid biocatalysts with peroxygenative activity that insert oxygen into non-activated compounds, while also possessing convergent peroxidative activity for one electron oxidation reactions. In several ligninolytic peroxidases, the site of peroxidative activity is associated with an oxidizable aromatic residue at the protein surface that connects to the buried heme domain through a long-range electron transfer (LRET) pathway. However, the peroxidative activity of these enzymes may also be initiated at the heme access channel. In this study, we examined the origin of the peroxidative activity of UPOs using an evolved secretion variant (PaDa-I mutant) from Agrocybe aegerita as our point of departure. After analyzing potential radical-forming aromatic residues at the PaDa-I surface by QM/MM, independent saturation mutagenesis libraries of Trp24, Tyr47, Tyr79, Tyr151, Tyr265, Tyr281, Tyr293 and Tyr325 were constructed and screened with both peroxidative and peroxygenative substrates. These mutant libraries were mostly inactive, with only a few functional clones detected, none of these showing marked differences in the peroxygenative and peroxidative activities. By contrast, when the flexible Gly314-Gly318 loop that is found at the outer entrance to the heme channel was subjected to combinatorial saturation mutagenesis and computational analysis, mutants with improved kinetics and a shift in the pH activity profile for peroxidative substrates were found, while they retained their kinetic values for peroxygenative substrates. This striking change was accompanied by a 4.5°C enhancement in kinetic thermostability despite the variants carried up to four consecutive mutations. Taken together, our study proves that the origin of the peroxidative activity in UPOs, unlike other ligninolytic peroxidases described to date, is not dependent on a LRET route from oxidizable residues at the protein surface, but rather it seems to be exclusively located at the heme access channel. Frontiers Media S.A. 2021-09-14 /pmc/articles/PMC8476742/ /pubmed/34595162 http://dx.doi.org/10.3389/fbioe.2021.741282 Text en Copyright © 2021 Molina-Espeja, Beltran-Nogal, Alfuzzi, Guallar and Alcalde. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Molina-Espeja, Patricia
Beltran-Nogal, Alejandro
Alfuzzi, Maria Alejandra
Guallar, Victor
Alcalde, Miguel
Mapping Potential Determinants of Peroxidative Activity in an Evolved Fungal Peroxygenase from Agrocybe aegerita
title Mapping Potential Determinants of Peroxidative Activity in an Evolved Fungal Peroxygenase from Agrocybe aegerita
title_full Mapping Potential Determinants of Peroxidative Activity in an Evolved Fungal Peroxygenase from Agrocybe aegerita
title_fullStr Mapping Potential Determinants of Peroxidative Activity in an Evolved Fungal Peroxygenase from Agrocybe aegerita
title_full_unstemmed Mapping Potential Determinants of Peroxidative Activity in an Evolved Fungal Peroxygenase from Agrocybe aegerita
title_short Mapping Potential Determinants of Peroxidative Activity in an Evolved Fungal Peroxygenase from Agrocybe aegerita
title_sort mapping potential determinants of peroxidative activity in an evolved fungal peroxygenase from agrocybe aegerita
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8476742/
https://www.ncbi.nlm.nih.gov/pubmed/34595162
http://dx.doi.org/10.3389/fbioe.2021.741282
work_keys_str_mv AT molinaespejapatricia mappingpotentialdeterminantsofperoxidativeactivityinanevolvedfungalperoxygenasefromagrocybeaegerita
AT beltrannogalalejandro mappingpotentialdeterminantsofperoxidativeactivityinanevolvedfungalperoxygenasefromagrocybeaegerita
AT alfuzzimariaalejandra mappingpotentialdeterminantsofperoxidativeactivityinanevolvedfungalperoxygenasefromagrocybeaegerita
AT guallarvictor mappingpotentialdeterminantsofperoxidativeactivityinanevolvedfungalperoxygenasefromagrocybeaegerita
AT alcaldemiguel mappingpotentialdeterminantsofperoxidativeactivityinanevolvedfungalperoxygenasefromagrocybeaegerita