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Fusion to Hydrophobin HFBI Improves the Catalytic Performance of a Cytochrome P450 System

Cytochrome P450 monooxygenases (P450) are heme-containing enzymes that oxidize a broad range of substrates in the presence of molecular oxygen and NAD(P)H. For their activity, most P450s rely on one or two redox proteins responsible for the transfer of electrons from the cofactor NAD(P)H to the heme...

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Autores principales: Schulz, Sebastian, Schumacher, Dominik, Raszkowski, Daniel, Girhard, Marco, Urlacher, Vlada B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4930934/
https://www.ncbi.nlm.nih.gov/pubmed/27458582
http://dx.doi.org/10.3389/fbioe.2016.00057
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author Schulz, Sebastian
Schumacher, Dominik
Raszkowski, Daniel
Girhard, Marco
Urlacher, Vlada B.
author_facet Schulz, Sebastian
Schumacher, Dominik
Raszkowski, Daniel
Girhard, Marco
Urlacher, Vlada B.
author_sort Schulz, Sebastian
collection PubMed
description Cytochrome P450 monooxygenases (P450) are heme-containing enzymes that oxidize a broad range of substrates in the presence of molecular oxygen and NAD(P)H. For their activity, most P450s rely on one or two redox proteins responsible for the transfer of electrons from the cofactor NAD(P)H to the heme. One of the challenges when using P450s in vitro, especially when non-physiological redox proteins are applied, is the inefficient transfer of electrons between the individual proteins resulting in non-productive consumption of NAD(P)H – referred to as uncoupling. Herein, we describe the improvement of the coupling efficiency between a P450 and its redox partner – diflavin reductase – by fusing both enzymes individually to the hydrophobin HFBI – a small self-assembling protein of the fungus Trichoderma reesei. The separated monooxygenase (BMO) and reductase (BMR) domains of P450 BM3 from Bacillus megaterium were chosen as a P450-reductase model system and individually fused to HFBI. The fusion proteins could be expressed in soluble form in Escherichia coli. When HFBI-fused BMO and BMR were mixed in vitro, substantially higher coupling efficiencies were measured as compared with the respective non-fused enzymes. Consequently, myristic acid conversion increased up to 20-fold (after 6 h) and 5-fold (after 24 h). Size exclusion chromatography demonstrated that in vitro the hydrophobin-fused enzymes build multimeric protein assemblies. Thus, the higher activity is hypothesized to be due to HFBI-mediated self-assembly arranging BMO and BMR in close spatial proximity in aqueous solution.
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spelling pubmed-49309342016-07-25 Fusion to Hydrophobin HFBI Improves the Catalytic Performance of a Cytochrome P450 System Schulz, Sebastian Schumacher, Dominik Raszkowski, Daniel Girhard, Marco Urlacher, Vlada B. Front Bioeng Biotechnol Bioengineering and Biotechnology Cytochrome P450 monooxygenases (P450) are heme-containing enzymes that oxidize a broad range of substrates in the presence of molecular oxygen and NAD(P)H. For their activity, most P450s rely on one or two redox proteins responsible for the transfer of electrons from the cofactor NAD(P)H to the heme. One of the challenges when using P450s in vitro, especially when non-physiological redox proteins are applied, is the inefficient transfer of electrons between the individual proteins resulting in non-productive consumption of NAD(P)H – referred to as uncoupling. Herein, we describe the improvement of the coupling efficiency between a P450 and its redox partner – diflavin reductase – by fusing both enzymes individually to the hydrophobin HFBI – a small self-assembling protein of the fungus Trichoderma reesei. The separated monooxygenase (BMO) and reductase (BMR) domains of P450 BM3 from Bacillus megaterium were chosen as a P450-reductase model system and individually fused to HFBI. The fusion proteins could be expressed in soluble form in Escherichia coli. When HFBI-fused BMO and BMR were mixed in vitro, substantially higher coupling efficiencies were measured as compared with the respective non-fused enzymes. Consequently, myristic acid conversion increased up to 20-fold (after 6 h) and 5-fold (after 24 h). Size exclusion chromatography demonstrated that in vitro the hydrophobin-fused enzymes build multimeric protein assemblies. Thus, the higher activity is hypothesized to be due to HFBI-mediated self-assembly arranging BMO and BMR in close spatial proximity in aqueous solution. Frontiers Media S.A. 2016-07-04 /pmc/articles/PMC4930934/ /pubmed/27458582 http://dx.doi.org/10.3389/fbioe.2016.00057 Text en Copyright © 2016 Schulz, Schumacher, Raszkowski, Girhard and Urlacher. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Schulz, Sebastian
Schumacher, Dominik
Raszkowski, Daniel
Girhard, Marco
Urlacher, Vlada B.
Fusion to Hydrophobin HFBI Improves the Catalytic Performance of a Cytochrome P450 System
title Fusion to Hydrophobin HFBI Improves the Catalytic Performance of a Cytochrome P450 System
title_full Fusion to Hydrophobin HFBI Improves the Catalytic Performance of a Cytochrome P450 System
title_fullStr Fusion to Hydrophobin HFBI Improves the Catalytic Performance of a Cytochrome P450 System
title_full_unstemmed Fusion to Hydrophobin HFBI Improves the Catalytic Performance of a Cytochrome P450 System
title_short Fusion to Hydrophobin HFBI Improves the Catalytic Performance of a Cytochrome P450 System
title_sort fusion to hydrophobin hfbi improves the catalytic performance of a cytochrome p450 system
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4930934/
https://www.ncbi.nlm.nih.gov/pubmed/27458582
http://dx.doi.org/10.3389/fbioe.2016.00057
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