Structural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA

Haloalkane dehalogenases (EC 3.8.1.5) play an important role in hydrolytic degradation of halogenated compounds, resulting in a halide ion, a proton, and an alcohol. They are used in biocatalysis, bioremediation, and biosensing of environmental pollutants and also for molecular tagging in cell biolo...

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Autores principales:	Andrii Mazur, Pavel Grinkevich, Radka Chaloupkova, Petra Havlickova, Barbora Kascakova, Michal Kuty, Jiri Damborsky, Ivana Kuta Smatanova, Tatyana Prudnikova
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	haloalkane dehalogenase ancestral sequence reconstruction structural analysis halogenated pollutants Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/0080554880414895a007514b52f70887
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spelling	oai:doaj.org-article:0080554880414895a007514b52f708872021-11-11T17:24:38ZStructural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA10.3390/ijms2221119921422-00671661-6596https://doaj.org/article/0080554880414895a007514b52f708872021-11-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/21/11992https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067Haloalkane dehalogenases (EC 3.8.1.5) play an important role in hydrolytic degradation of halogenated compounds, resulting in a halide ion, a proton, and an alcohol. They are used in biocatalysis, bioremediation, and biosensing of environmental pollutants and also for molecular tagging in cell biology. The method of ancestral sequence reconstruction leads to prediction of sequences of ancestral enzymes allowing their experimental characterization. Based on the sequences of modern haloalkane dehalogenases from the subfamily II, the most common ancestor of thoroughly characterized enzymes LinB from <i>Sphingobium japonicum</i> UT26 and DmbA from <i>Mycobacterium bovis</i> 5033/66 was in silico predicted, recombinantly produced and structurally characterized. The ancestral enzyme AncLinB-DmbA was crystallized using the sitting-drop vapor-diffusion method, yielding rod-like crystals that diffracted X-rays to 1.5 Å resolution. Structural comparison of AncLinB-DmbA with their closely related descendants LinB and DmbA revealed some differences in overall structure and tunnel architecture. Newly prepared AncLinB-DmbA has the highest active site cavity volume and the biggest entrance radius on the main tunnel in comparison to descendant enzymes. Ancestral sequence reconstruction is a powerful technique to study molecular evolution and design robust proteins for enzyme technologies.Andrii MazurPavel GrinkevichRadka ChaloupkovaPetra HavlickovaBarbora KascakovaMichal KutyJiri DamborskyIvana Kuta SmatanovaTatyana PrudnikovaMDPI AGarticlehaloalkane dehalogenaseancestral sequence reconstructionstructural analysishalogenated pollutantsBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 11992, p 11992 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	haloalkane dehalogenase ancestral sequence reconstruction structural analysis halogenated pollutants Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	haloalkane dehalogenase ancestral sequence reconstruction structural analysis halogenated pollutants Biology (General) QH301-705.5 Chemistry QD1-999 Andrii Mazur Pavel Grinkevich Radka Chaloupkova Petra Havlickova Barbora Kascakova Michal Kuty Jiri Damborsky Ivana Kuta Smatanova Tatyana Prudnikova Structural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA
description	Haloalkane dehalogenases (EC 3.8.1.5) play an important role in hydrolytic degradation of halogenated compounds, resulting in a halide ion, a proton, and an alcohol. They are used in biocatalysis, bioremediation, and biosensing of environmental pollutants and also for molecular tagging in cell biology. The method of ancestral sequence reconstruction leads to prediction of sequences of ancestral enzymes allowing their experimental characterization. Based on the sequences of modern haloalkane dehalogenases from the subfamily II, the most common ancestor of thoroughly characterized enzymes LinB from <i>Sphingobium japonicum</i> UT26 and DmbA from <i>Mycobacterium bovis</i> 5033/66 was in silico predicted, recombinantly produced and structurally characterized. The ancestral enzyme AncLinB-DmbA was crystallized using the sitting-drop vapor-diffusion method, yielding rod-like crystals that diffracted X-rays to 1.5 Å resolution. Structural comparison of AncLinB-DmbA with their closely related descendants LinB and DmbA revealed some differences in overall structure and tunnel architecture. Newly prepared AncLinB-DmbA has the highest active site cavity volume and the biggest entrance radius on the main tunnel in comparison to descendant enzymes. Ancestral sequence reconstruction is a powerful technique to study molecular evolution and design robust proteins for enzyme technologies.
format	article
author	Andrii Mazur Pavel Grinkevich Radka Chaloupkova Petra Havlickova Barbora Kascakova Michal Kuty Jiri Damborsky Ivana Kuta Smatanova Tatyana Prudnikova
author_facet	Andrii Mazur Pavel Grinkevich Radka Chaloupkova Petra Havlickova Barbora Kascakova Michal Kuty Jiri Damborsky Ivana Kuta Smatanova Tatyana Prudnikova
author_sort	Andrii Mazur
title	Structural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA
title_short	Structural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA
title_full	Structural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA
title_fullStr	Structural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA
title_full_unstemmed	Structural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA
title_sort	structural analysis of the ancestral haloalkane dehalogenase anclinb-dmba
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/0080554880414895a007514b52f70887
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Structural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA

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