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Lysine Decarboxylase with an Enhanced Affinity for Pyridoxal 5-Phosphate by Disulfide Bond-Mediated Spatial Reconstitution

Lysine decarboxylase (LDC) catalyzes the decarboxylation of l-lysine to produce cadaverine, an important industrial platform chemical for bio-based polyamides. However, due to high flexibility at the pyridoxal 5-phosphate (PLP) binding site, use of the enzyme for cadaverine production requires conti...

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Autores principales: Sagong, Hye-Young, Kim, Kyung-Jin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240995/
https://www.ncbi.nlm.nih.gov/pubmed/28095457
http://dx.doi.org/10.1371/journal.pone.0170163
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author Sagong, Hye-Young
Kim, Kyung-Jin
author_facet Sagong, Hye-Young
Kim, Kyung-Jin
author_sort Sagong, Hye-Young
collection PubMed
description Lysine decarboxylase (LDC) catalyzes the decarboxylation of l-lysine to produce cadaverine, an important industrial platform chemical for bio-based polyamides. However, due to high flexibility at the pyridoxal 5-phosphate (PLP) binding site, use of the enzyme for cadaverine production requires continuous supplement of large amounts of PLP. In order to develop an LDC enzyme from Selenomonas ruminantium (SrLDC) with an enhanced affinity for PLP, we introduced an internal disulfide bond between Ala225 and Thr302 residues with a desire to retain the PLP binding site in a closed conformation. The SrLDC(A225C/T302C) mutant showed a yellow color and the characteristic UV/Vis absorption peaks for enzymes with bound PLP, and exhibited three-fold enhanced PLP affinity compared with the wild-type SrLDC. The mutant also exhibited a dramatically enhanced LDC activity and cadaverine conversion particularly under no or low PLP concentrations. Moreover, introduction of the disulfide bond rendered SrLDC more resistant to high pH and temperature. The formation of the introduced disulfide bond and the maintenance of the PLP binding site in the closed conformation were confirmed by determination of the crystal structure of the mutant. This study shows that disulfide bond-mediated spatial reconstitution can be a platform technology for development of enzymes with enhanced PLP affinity.
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spelling pubmed-52409952017-02-06 Lysine Decarboxylase with an Enhanced Affinity for Pyridoxal 5-Phosphate by Disulfide Bond-Mediated Spatial Reconstitution Sagong, Hye-Young Kim, Kyung-Jin PLoS One Research Article Lysine decarboxylase (LDC) catalyzes the decarboxylation of l-lysine to produce cadaverine, an important industrial platform chemical for bio-based polyamides. However, due to high flexibility at the pyridoxal 5-phosphate (PLP) binding site, use of the enzyme for cadaverine production requires continuous supplement of large amounts of PLP. In order to develop an LDC enzyme from Selenomonas ruminantium (SrLDC) with an enhanced affinity for PLP, we introduced an internal disulfide bond between Ala225 and Thr302 residues with a desire to retain the PLP binding site in a closed conformation. The SrLDC(A225C/T302C) mutant showed a yellow color and the characteristic UV/Vis absorption peaks for enzymes with bound PLP, and exhibited three-fold enhanced PLP affinity compared with the wild-type SrLDC. The mutant also exhibited a dramatically enhanced LDC activity and cadaverine conversion particularly under no or low PLP concentrations. Moreover, introduction of the disulfide bond rendered SrLDC more resistant to high pH and temperature. The formation of the introduced disulfide bond and the maintenance of the PLP binding site in the closed conformation were confirmed by determination of the crystal structure of the mutant. This study shows that disulfide bond-mediated spatial reconstitution can be a platform technology for development of enzymes with enhanced PLP affinity. Public Library of Science 2017-01-17 /pmc/articles/PMC5240995/ /pubmed/28095457 http://dx.doi.org/10.1371/journal.pone.0170163 Text en © 2017 Sagong, Kim http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Sagong, Hye-Young
Kim, Kyung-Jin
Lysine Decarboxylase with an Enhanced Affinity for Pyridoxal 5-Phosphate by Disulfide Bond-Mediated Spatial Reconstitution
title Lysine Decarboxylase with an Enhanced Affinity for Pyridoxal 5-Phosphate by Disulfide Bond-Mediated Spatial Reconstitution
title_full Lysine Decarboxylase with an Enhanced Affinity for Pyridoxal 5-Phosphate by Disulfide Bond-Mediated Spatial Reconstitution
title_fullStr Lysine Decarboxylase with an Enhanced Affinity for Pyridoxal 5-Phosphate by Disulfide Bond-Mediated Spatial Reconstitution
title_full_unstemmed Lysine Decarboxylase with an Enhanced Affinity for Pyridoxal 5-Phosphate by Disulfide Bond-Mediated Spatial Reconstitution
title_short Lysine Decarboxylase with an Enhanced Affinity for Pyridoxal 5-Phosphate by Disulfide Bond-Mediated Spatial Reconstitution
title_sort lysine decarboxylase with an enhanced affinity for pyridoxal 5-phosphate by disulfide bond-mediated spatial reconstitution
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240995/
https://www.ncbi.nlm.nih.gov/pubmed/28095457
http://dx.doi.org/10.1371/journal.pone.0170163
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