Engineering the Catalytic Properties of Two-Domain Laccase from <i>Streptomyces griseoflavus</i> 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 <i>Streptomyce...
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MDPI AG
2021-12-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/1/65 |
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| author | Ilya Kolyadenko Anastasia Scherbakova Kirill Kovalev Azat Gabdulkhakov Svetlana Tishchenko |
| author_facet | Ilya Kolyadenko Anastasia Scherbakova Kirill Kovalev Azat Gabdulkhakov Svetlana Tishchenko |
| author_sort | Ilya Kolyadenko |
| collection | DOAJ |
| 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 <i>Streptomyces griseoflavus</i> (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. |
| first_indexed | 2024-03-10T03:41:06Z |
| format | Article |
| id | doaj.art-fbc9251238c648dcb8bf159e7c217553 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T03:41:06Z |
| publishDate | 2021-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-fbc9251238c648dcb8bf159e7c2175532023-11-23T11:34:08ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-12-012316510.3390/ijms23010065Engineering the Catalytic Properties of Two-Domain Laccase from <i>Streptomyces griseoflavus</i> Ac-993Ilya Kolyadenko0Anastasia Scherbakova1Kirill Kovalev2Azat Gabdulkhakov3Svetlana Tishchenko4Institute of Protein Research RAS, 142290 Pushchino, RussiaInstitute of Protein Research RAS, 142290 Pushchino, RussiaEuropean Molecular Biology Laboratory, 22607 Hamburg, GermanyInstitute of Protein Research RAS, 142290 Pushchino, RussiaInstitute of Protein Research RAS, 142290 Pushchino, RussiaLaccases 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 <i>Streptomyces griseoflavus</i> (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.https://www.mdpi.com/1422-0067/23/1/65two-domain laccasescrystal structuressite-directed mutagenesisproton wiresubstrate-binding sitecatalytic activity |
| spellingShingle | Ilya Kolyadenko Anastasia Scherbakova Kirill Kovalev Azat Gabdulkhakov Svetlana Tishchenko Engineering the Catalytic Properties of Two-Domain Laccase from <i>Streptomyces griseoflavus</i> Ac-993 International Journal of Molecular Sciences two-domain laccases crystal structures site-directed mutagenesis proton wire substrate-binding site catalytic activity |
| title | Engineering the Catalytic Properties of Two-Domain Laccase from <i>Streptomyces griseoflavus</i> Ac-993 |
| title_full | Engineering the Catalytic Properties of Two-Domain Laccase from <i>Streptomyces griseoflavus</i> Ac-993 |
| title_fullStr | Engineering the Catalytic Properties of Two-Domain Laccase from <i>Streptomyces griseoflavus</i> Ac-993 |
| title_full_unstemmed | Engineering the Catalytic Properties of Two-Domain Laccase from <i>Streptomyces griseoflavus</i> Ac-993 |
| title_short | Engineering the Catalytic Properties of Two-Domain Laccase from <i>Streptomyces griseoflavus</i> Ac-993 |
| title_sort | engineering the catalytic properties of two domain laccase from i streptomyces griseoflavus i ac 993 |
| topic | two-domain laccases crystal structures site-directed mutagenesis proton wire substrate-binding site catalytic activity |
| url | https://www.mdpi.com/1422-0067/23/1/65 |
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