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Engineering the Catalytic Properties of Two-Domain Laccase from Streptomyces griseoflavus Ac-993

Laccases catalyze the oxidation of substrates with the concomitant reduction of oxygen to water. Recently, we found that polar residues located in tunnels leading to Cu2 and Cu3 ions control oxygen entrance (His 165) and proton transport (Arg 240) of two-domain laccase (2D) from Streptomyces griseof...

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Autores principales: Kolyadenko, Ilya, Scherbakova, Anastasia, Kovalev, Kirill, Gabdulkhakov, Azat, Tishchenko, Svetlana
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8744557/
https://www.ncbi.nlm.nih.gov/pubmed/35008493
http://dx.doi.org/10.3390/ijms23010065
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author Kolyadenko, Ilya
Scherbakova, Anastasia
Kovalev, Kirill
Gabdulkhakov, Azat
Tishchenko, Svetlana
author_facet Kolyadenko, Ilya
Scherbakova, Anastasia
Kovalev, Kirill
Gabdulkhakov, Azat
Tishchenko, Svetlana
author_sort Kolyadenko, Ilya
collection PubMed
description Laccases catalyze the oxidation of substrates with the concomitant reduction of oxygen to water. Recently, we found that polar residues located in tunnels leading to Cu2 and Cu3 ions control oxygen entrance (His 165) and proton transport (Arg 240) of two-domain laccase (2D) from Streptomyces griseoflavus (SgfSL). In this work, we have focused on optimizing the substrate-binding pocket (SBP) of SgfSL while simultaneously adjusting the oxygen reduction process. SgfSL variants with three single (Met199Ala, Met199Gly, and Tyr230Ala) and three double amino acid residues substitutions (Met199Gly/His165Ala, His165Ala/Arg240His, Met199Gly/Arg240His) were constructed, purified, and investigated. Combination of substitutions in the SBP and in the tunnel leading to Cu2 ion (Met199Gly/Arg240His) increased SgfSL catalytic activity towards ABTS by 5-fold, and towards 2.6-DMP by 16-fold. The high activity of the Met199Gly/Arg240His variant can be explained by the combined effect of the SBP geometry optimization (Met199Gly) and increased proton flux via the tunnel leading to Cu2 ion (Arg240His). Moreover, the variant with Met199Gly and His165Ala mutations did not significantly increase SgfSL’s activity, but led to a drastic shift in the optimal pH of 2.6-DMP oxidation. These results indicate that His 165 not only regulates oxygen access, but it also participates in proton transport in 2D laccases.
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spelling pubmed-87445572022-01-11 Engineering the Catalytic Properties of Two-Domain Laccase from Streptomyces griseoflavus Ac-993 Kolyadenko, Ilya Scherbakova, Anastasia Kovalev, Kirill Gabdulkhakov, Azat Tishchenko, Svetlana Int J Mol Sci Article Laccases catalyze the oxidation of substrates with the concomitant reduction of oxygen to water. Recently, we found that polar residues located in tunnels leading to Cu2 and Cu3 ions control oxygen entrance (His 165) and proton transport (Arg 240) of two-domain laccase (2D) from Streptomyces griseoflavus (SgfSL). In this work, we have focused on optimizing the substrate-binding pocket (SBP) of SgfSL while simultaneously adjusting the oxygen reduction process. SgfSL variants with three single (Met199Ala, Met199Gly, and Tyr230Ala) and three double amino acid residues substitutions (Met199Gly/His165Ala, His165Ala/Arg240His, Met199Gly/Arg240His) were constructed, purified, and investigated. Combination of substitutions in the SBP and in the tunnel leading to Cu2 ion (Met199Gly/Arg240His) increased SgfSL catalytic activity towards ABTS by 5-fold, and towards 2.6-DMP by 16-fold. The high activity of the Met199Gly/Arg240His variant can be explained by the combined effect of the SBP geometry optimization (Met199Gly) and increased proton flux via the tunnel leading to Cu2 ion (Arg240His). Moreover, the variant with Met199Gly and His165Ala mutations did not significantly increase SgfSL’s activity, but led to a drastic shift in the optimal pH of 2.6-DMP oxidation. These results indicate that His 165 not only regulates oxygen access, but it also participates in proton transport in 2D laccases. MDPI 2021-12-22 /pmc/articles/PMC8744557/ /pubmed/35008493 http://dx.doi.org/10.3390/ijms23010065 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Kolyadenko, Ilya
Scherbakova, Anastasia
Kovalev, Kirill
Gabdulkhakov, Azat
Tishchenko, Svetlana
Engineering the Catalytic Properties of Two-Domain Laccase from Streptomyces griseoflavus Ac-993
title Engineering the Catalytic Properties of Two-Domain Laccase from Streptomyces griseoflavus Ac-993
title_full Engineering the Catalytic Properties of Two-Domain Laccase from Streptomyces griseoflavus Ac-993
title_fullStr Engineering the Catalytic Properties of Two-Domain Laccase from Streptomyces griseoflavus Ac-993
title_full_unstemmed Engineering the Catalytic Properties of Two-Domain Laccase from Streptomyces griseoflavus Ac-993
title_short Engineering the Catalytic Properties of Two-Domain Laccase from Streptomyces griseoflavus Ac-993
title_sort engineering the catalytic properties of two-domain laccase from streptomyces griseoflavus ac-993
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8744557/
https://www.ncbi.nlm.nih.gov/pubmed/35008493
http://dx.doi.org/10.3390/ijms23010065
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