Structural inspection and protein motions modelling of a fungal glycoside hydrolase family 18 chitinase by crystallography depicts a dynamic enzymatic mechanism
Chitinases degrade chitin into low molecular weight chitooligomers, which have a broad range of industrial, agricultural, and medical functions. Understanding the relationship between the diverse characteristics of chitinases and their functions is necessary for the improvement of functional enzymes...
| Autores: | , , , |
|---|---|
| Tipo de recurso: | artículo |
| Fecha de publicación: | 2021 |
| País: | España |
| Institución: | Universidad Autónoma de Madrid |
| Repositorio: | Biblos-e Archivo. Repositorio Institucional de la UAM |
| Idioma: | inglés |
| OAI Identifier: | oai:repositorio.uam.es:10486/716543 |
| Acceso en línea: | http://hdl.handle.net/10486/716543 https://dx.doi.org/10.1016/j.csbj.2021.09.027 |
| Access Level: | acceso abierto |
| Palabra clave: | Chitinase crystal structure chitin oligosaccharide binding mode dynamic mechanism specificity Biología y Biomedicina / Biología |
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Structural inspection and protein motions modelling of a fungal glycoside hydrolase family 18 chitinase by crystallography depicts a dynamic enzymatic mechanismJiménez-Ortega, ElenaKidibule, Peter EliasFernández Lobato, MaríaSanz‑Aparicio, JuliaChitinasecrystal structurechitin oligosaccharidebinding modedynamic mechanismspecificityBiología y Biomedicina / BiologíaChitinases degrade chitin into low molecular weight chitooligomers, which have a broad range of industrial, agricultural, and medical functions. Understanding the relationship between the diverse characteristics of chitinases and their functions is necessary for the improvement of functional enzymes that meet specific requirements. We report here a full crystallographic analysis of three complexes obtained from the chitinase Chit42 from Trichoderma harzianum, which represent different states along the enzymatic mechanism. The inactive double mutant D169A/E171A was submitted to soaking/crystallization experiments with hexa-N-acetyl-glucosamine (NAG6) or tetra-N-acetyl-glucosamine (NAG4), trapping the enzyme-substrate complex (Chit42-NAG6), the enzyme-products complex (Chit42-NAG4-NAG2) and a someway intermediate state. Structural comparison among the different complexes depicts the determinants defining the different subsites and revealed a previously unobserved dynamic on-off ligand binding process associated with a motion of its insertion domain, which might be accompanying the role or aromatics in processivity. An ensemble refinement performed to extract dynamic details from the diffraction data elucidates the implication of some highly flexible residues in the productive sliding of the substrate and the product release event. These positions were submitted to mutagenesis and the activity of the variants was investigated in the hydrolysis of NAG6, colloidal chitin and two chitosans with different polymerization and acetylation degree. All the changes affected the Chit42 hydrolytic activity therefore confirming the involvement of these positions in catalysis. Furthermore, we found the variants R295S and E316S improving the apparent catalytic efficiency of chitin and NAG6 and, together with E316A, enhancing the specific activity on chitosan. Therefore, our results provide novel insight into the molecular mechanisms underlying the hydrolysis of chitinous material by fungal chitinases, and suggest new targets to address engineering of these biotechnologically important enzymesThis work was supported by grants from the Spanish Ministry of Economy and Competitiveness through grants BIO2016‐76601‐C3‐3‐R/-C3-2-R, PID2019-105838RB-C33/-C32, Fundación Ramón Areces [XIX Call of Research Grants in Life and Material Sciences] and EU EMFF-Blue Economy-2018 [Fish4Fish-863697]. We are grateful to the staff of the Synchrotron Radiation Sources at Alba (Barcelona, Spain) for providing access and for technical assistance at BL13‐XALOC beamline and to the Fundación Ramón Areces for an institutional grant to the Centre of Molecular Biology Severo OchoaElsevier B.V. on behalf of Research Network of Computational and Structural BiotechnologyDepartamento de Biología MolecularFacultad de Ciencias20212021-10-02research articlehttp://purl.org/coar/resource_type/c_2df8fbb1VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10486/716543https://dx.doi.org/10.1016/j.csbj.2021.09.027reponame:Biblos-e Archivo. Repositorio Institucional de la UAMinstname:Universidad Autónoma de MadridInglésengopen accesshttp://purl.org/coar/access_right/c_abf2Attribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessoai:repositorio.uam.es:10486/7165432026-06-23T12:46:27Z |
| dc.title.none.fl_str_mv |
Structural inspection and protein motions modelling of a fungal glycoside hydrolase family 18 chitinase by crystallography depicts a dynamic enzymatic mechanism |
| title |
Structural inspection and protein motions modelling of a fungal glycoside hydrolase family 18 chitinase by crystallography depicts a dynamic enzymatic mechanism |
| spellingShingle |
Structural inspection and protein motions modelling of a fungal glycoside hydrolase family 18 chitinase by crystallography depicts a dynamic enzymatic mechanism Jiménez-Ortega, Elena Chitinase crystal structure chitin oligosaccharide binding mode dynamic mechanism specificity Biología y Biomedicina / Biología |
| title_short |
Structural inspection and protein motions modelling of a fungal glycoside hydrolase family 18 chitinase by crystallography depicts a dynamic enzymatic mechanism |
| title_full |
Structural inspection and protein motions modelling of a fungal glycoside hydrolase family 18 chitinase by crystallography depicts a dynamic enzymatic mechanism |
| title_fullStr |
Structural inspection and protein motions modelling of a fungal glycoside hydrolase family 18 chitinase by crystallography depicts a dynamic enzymatic mechanism |
| title_full_unstemmed |
Structural inspection and protein motions modelling of a fungal glycoside hydrolase family 18 chitinase by crystallography depicts a dynamic enzymatic mechanism |
| title_sort |
Structural inspection and protein motions modelling of a fungal glycoside hydrolase family 18 chitinase by crystallography depicts a dynamic enzymatic mechanism |
| dc.creator.none.fl_str_mv |
Jiménez-Ortega, Elena Kidibule, Peter Elias Fernández Lobato, María Sanz‑Aparicio, Julia |
| author |
Jiménez-Ortega, Elena |
| author_facet |
Jiménez-Ortega, Elena Kidibule, Peter Elias Fernández Lobato, María Sanz‑Aparicio, Julia |
| author_role |
author |
| author2 |
Kidibule, Peter Elias Fernández Lobato, María Sanz‑Aparicio, Julia |
| author2_role |
author author author |
| dc.contributor.none.fl_str_mv |
Departamento de Biología Molecular Facultad de Ciencias |
| dc.subject.none.fl_str_mv |
Chitinase crystal structure chitin oligosaccharide binding mode dynamic mechanism specificity Biología y Biomedicina / Biología |
| topic |
Chitinase crystal structure chitin oligosaccharide binding mode dynamic mechanism specificity Biología y Biomedicina / Biología |
| description |
Chitinases degrade chitin into low molecular weight chitooligomers, which have a broad range of industrial, agricultural, and medical functions. Understanding the relationship between the diverse characteristics of chitinases and their functions is necessary for the improvement of functional enzymes that meet specific requirements. We report here a full crystallographic analysis of three complexes obtained from the chitinase Chit42 from Trichoderma harzianum, which represent different states along the enzymatic mechanism. The inactive double mutant D169A/E171A was submitted to soaking/crystallization experiments with hexa-N-acetyl-glucosamine (NAG6) or tetra-N-acetyl-glucosamine (NAG4), trapping the enzyme-substrate complex (Chit42-NAG6), the enzyme-products complex (Chit42-NAG4-NAG2) and a someway intermediate state. Structural comparison among the different complexes depicts the determinants defining the different subsites and revealed a previously unobserved dynamic on-off ligand binding process associated with a motion of its insertion domain, which might be accompanying the role or aromatics in processivity. An ensemble refinement performed to extract dynamic details from the diffraction data elucidates the implication of some highly flexible residues in the productive sliding of the substrate and the product release event. These positions were submitted to mutagenesis and the activity of the variants was investigated in the hydrolysis of NAG6, colloidal chitin and two chitosans with different polymerization and acetylation degree. All the changes affected the Chit42 hydrolytic activity therefore confirming the involvement of these positions in catalysis. Furthermore, we found the variants R295S and E316S improving the apparent catalytic efficiency of chitin and NAG6 and, together with E316A, enhancing the specific activity on chitosan. Therefore, our results provide novel insight into the molecular mechanisms underlying the hydrolysis of chitinous material by fungal chitinases, and suggest new targets to address engineering of these biotechnologically important enzymes |
| publishDate |
2021 |
| dc.date.none.fl_str_mv |
2021 2021-10-02 |
| dc.type.none.fl_str_mv |
research article http://purl.org/coar/resource_type/c_2df8fbb1 VoR http://purl.org/coar/version/c_970fb48d4fbd8a85 |
| dc.type.openaire.fl_str_mv |
info:eu-repo/semantics/article |
| format |
article |
| dc.identifier.none.fl_str_mv |
http://hdl.handle.net/10486/716543 https://dx.doi.org/10.1016/j.csbj.2021.09.027 |
| url |
http://hdl.handle.net/10486/716543 https://dx.doi.org/10.1016/j.csbj.2021.09.027 |
| dc.language.none.fl_str_mv |
Inglés eng |
| language_invalid_str_mv |
Inglés |
| language |
eng |
| dc.rights.none.fl_str_mv |
open access http://purl.org/coar/access_right/c_abf2 Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ |
| dc.rights.openaire.fl_str_mv |
info:eu-repo/semantics/openAccess |
| rights_invalid_str_mv |
open access http://purl.org/coar/access_right/c_abf2 Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ |
| eu_rights_str_mv |
openAccess |
| dc.format.none.fl_str_mv |
application/pdf |
| dc.publisher.none.fl_str_mv |
Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology |
| publisher.none.fl_str_mv |
Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology |
| dc.source.none.fl_str_mv |
reponame:Biblos-e Archivo. Repositorio Institucional de la UAM instname:Universidad Autónoma de Madrid |
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Universidad Autónoma de Madrid |
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Biblos-e Archivo. Repositorio Institucional de la UAM |
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Biblos-e Archivo. Repositorio Institucional de la UAM |
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