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Characterization of a Self-sufficient Trans-Anethole Oxygenase from Pseudomonas putida JYR-1

A novel flavoprotein monooxygenase, trans-anethole oxygenase (TAO), from Pseudomonas putida JYR-1, which is capable of catalyzing the oxidation of trans-anethole to p-anisaldehyde, was heterologously expressed in E. coli and purified. Enzymatic kinetics of diverse substrates and cofactors revealed t...

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Autores principales: Han, Dongfei, Sadowsky, Michael J., Chong, Youhoon, Hur, Hor-Gil
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774712/
https://www.ncbi.nlm.nih.gov/pubmed/24066043
http://dx.doi.org/10.1371/journal.pone.0073350
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author Han, Dongfei
Sadowsky, Michael J.
Chong, Youhoon
Hur, Hor-Gil
author_facet Han, Dongfei
Sadowsky, Michael J.
Chong, Youhoon
Hur, Hor-Gil
author_sort Han, Dongfei
collection PubMed
description A novel flavoprotein monooxygenase, trans-anethole oxygenase (TAO), from Pseudomonas putida JYR-1, which is capable of catalyzing the oxidation of trans-anethole to p-anisaldehyde, was heterologously expressed in E. coli and purified. Enzymatic kinetics of diverse substrates and cofactors revealed that TAO is likely to be a novel self-sufficient flavoprotein monooxygenase. Enzyme assays of GST-TAO demonstrated that TAO catalyzed a trans-anethole oxidation reaction without auxiliary component enzyme-like electron-transfer flavin reductases. The single component TAO had the ability to reduce flavin cofactors and simultaneously oxidize trans-anthole to p-anisaldehyde. In the processes of reduction of flavin and oxidation of trans-anethole, TAO accepted various flavin and NAD(P)H cofactors. TAO also catalyzed oxidation of isoeugenol, O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure with different catalytic efficiency. TAO had the greatest catalytic efficiency (k (cat)/K (m)) with the original substrate, trans-anethole. Investigation about partially deleted mutants of TAO indicated that reductase active sites appeared to be located near the N terminal. Site directed mutagenesis studies also proved that the proposed flavin binding sites, Trp-38, Thr-43, Tyr-55, were critical for flavin reduction. However, disruption of any portion of TAO eliminated the oxygenase activity.
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spelling pubmed-37747122013-09-24 Characterization of a Self-sufficient Trans-Anethole Oxygenase from Pseudomonas putida JYR-1 Han, Dongfei Sadowsky, Michael J. Chong, Youhoon Hur, Hor-Gil PLoS One Research Article A novel flavoprotein monooxygenase, trans-anethole oxygenase (TAO), from Pseudomonas putida JYR-1, which is capable of catalyzing the oxidation of trans-anethole to p-anisaldehyde, was heterologously expressed in E. coli and purified. Enzymatic kinetics of diverse substrates and cofactors revealed that TAO is likely to be a novel self-sufficient flavoprotein monooxygenase. Enzyme assays of GST-TAO demonstrated that TAO catalyzed a trans-anethole oxidation reaction without auxiliary component enzyme-like electron-transfer flavin reductases. The single component TAO had the ability to reduce flavin cofactors and simultaneously oxidize trans-anthole to p-anisaldehyde. In the processes of reduction of flavin and oxidation of trans-anethole, TAO accepted various flavin and NAD(P)H cofactors. TAO also catalyzed oxidation of isoeugenol, O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure with different catalytic efficiency. TAO had the greatest catalytic efficiency (k (cat)/K (m)) with the original substrate, trans-anethole. Investigation about partially deleted mutants of TAO indicated that reductase active sites appeared to be located near the N terminal. Site directed mutagenesis studies also proved that the proposed flavin binding sites, Trp-38, Thr-43, Tyr-55, were critical for flavin reduction. However, disruption of any portion of TAO eliminated the oxygenase activity. Public Library of Science 2013-09-16 /pmc/articles/PMC3774712/ /pubmed/24066043 http://dx.doi.org/10.1371/journal.pone.0073350 Text en © 2013 Han et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Han, Dongfei
Sadowsky, Michael J.
Chong, Youhoon
Hur, Hor-Gil
Characterization of a Self-sufficient Trans-Anethole Oxygenase from Pseudomonas putida JYR-1
title Characterization of a Self-sufficient Trans-Anethole Oxygenase from Pseudomonas putida JYR-1
title_full Characterization of a Self-sufficient Trans-Anethole Oxygenase from Pseudomonas putida JYR-1
title_fullStr Characterization of a Self-sufficient Trans-Anethole Oxygenase from Pseudomonas putida JYR-1
title_full_unstemmed Characterization of a Self-sufficient Trans-Anethole Oxygenase from Pseudomonas putida JYR-1
title_short Characterization of a Self-sufficient Trans-Anethole Oxygenase from Pseudomonas putida JYR-1
title_sort characterization of a self-sufficient trans-anethole oxygenase from pseudomonas putida jyr-1
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774712/
https://www.ncbi.nlm.nih.gov/pubmed/24066043
http://dx.doi.org/10.1371/journal.pone.0073350
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