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Conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis
Polyphenol oxidases (PPOs) comprise tyrosinases (TYRs) and catechol oxidases (COs), which catalyse the initial reactions in the biosynthesis of melanin. TYRs hydroxylate monophenolic (monophenolase activity) and oxidize diphenolic (diphenolase activity) substrates, whereas COs react only with diphen...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Nature Publishing Group UK
2020
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997208/ https://www.ncbi.nlm.nih.gov/pubmed/32015350 http://dx.doi.org/10.1038/s41598-020-57671-x |
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author | Panis, Felix Kampatsikas, Ioannis Bijelic, Aleksandar Rompel, Annette |
author_facet | Panis, Felix Kampatsikas, Ioannis Bijelic, Aleksandar Rompel, Annette |
author_sort | Panis, Felix |
collection | PubMed |
description | Polyphenol oxidases (PPOs) comprise tyrosinases (TYRs) and catechol oxidases (COs), which catalyse the initial reactions in the biosynthesis of melanin. TYRs hydroxylate monophenolic (monophenolase activity) and oxidize diphenolic (diphenolase activity) substrates, whereas COs react only with diphenols. In order to elucidate the biochemical basis for the different reactions in PPOs, cDNA from walnut leaves was synthesized, the target gene encoding the latent walnut tyrosinase (jrPPO1) was cloned, and the enzyme was heterologously expressed in Escherichia coli. Mutations targeting the two activity controller residues (Asn240 and Leu244) as well as the gatekeeper residue (Phe260) were designed to impair monophenolase activity of jrPPO1. For the first time, monophenolase activity of jrPPO1 towards L-tyrosine was blocked in two double mutants (Asn240Lys/Leu244Arg and Asn240Thr/Leu244Arg) while its diphenolase activity was partially preserved, thereby converting jrPPO1 into a CO. Kinetic data show that recombinant jrPPO1 resembles the natural enzyme, and spectrophotometric investigations proved that the copper content remains unaffected by the mutations. The results presented herein provide experimental evidence that a precisely tuned interplay between the amino acids located around the active center controls the substrate specificity and therewith the mono- versus diphenolase activity in the type-III copper enzyme jrPPO1. |
format | Online Article Text |
id | pubmed-6997208 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-69972082020-02-10 Conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis Panis, Felix Kampatsikas, Ioannis Bijelic, Aleksandar Rompel, Annette Sci Rep Article Polyphenol oxidases (PPOs) comprise tyrosinases (TYRs) and catechol oxidases (COs), which catalyse the initial reactions in the biosynthesis of melanin. TYRs hydroxylate monophenolic (monophenolase activity) and oxidize diphenolic (diphenolase activity) substrates, whereas COs react only with diphenols. In order to elucidate the biochemical basis for the different reactions in PPOs, cDNA from walnut leaves was synthesized, the target gene encoding the latent walnut tyrosinase (jrPPO1) was cloned, and the enzyme was heterologously expressed in Escherichia coli. Mutations targeting the two activity controller residues (Asn240 and Leu244) as well as the gatekeeper residue (Phe260) were designed to impair monophenolase activity of jrPPO1. For the first time, monophenolase activity of jrPPO1 towards L-tyrosine was blocked in two double mutants (Asn240Lys/Leu244Arg and Asn240Thr/Leu244Arg) while its diphenolase activity was partially preserved, thereby converting jrPPO1 into a CO. Kinetic data show that recombinant jrPPO1 resembles the natural enzyme, and spectrophotometric investigations proved that the copper content remains unaffected by the mutations. The results presented herein provide experimental evidence that a precisely tuned interplay between the amino acids located around the active center controls the substrate specificity and therewith the mono- versus diphenolase activity in the type-III copper enzyme jrPPO1. Nature Publishing Group UK 2020-02-03 /pmc/articles/PMC6997208/ /pubmed/32015350 http://dx.doi.org/10.1038/s41598-020-57671-x Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Panis, Felix Kampatsikas, Ioannis Bijelic, Aleksandar Rompel, Annette Conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis |
title | Conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis |
title_full | Conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis |
title_fullStr | Conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis |
title_full_unstemmed | Conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis |
title_short | Conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis |
title_sort | conversion of walnut tyrosinase into a catechol oxidase by site directed mutagenesis |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997208/ https://www.ncbi.nlm.nih.gov/pubmed/32015350 http://dx.doi.org/10.1038/s41598-020-57671-x |
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