Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity

Abstract Fusion of multiple enzymes to multifunctional constructs has been recognized as a viable strategy to improve enzymatic properties at various levels such as stability, activity and handling. In this study, the genes coding for cytochrome P450 BM3 from B. megaterium and formate dehydrogenase...

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Autores principales:	Arsenij Kokorin, Pavel D. Parshin, Patrick J. Bakkes, Anastasia A. Pometun, Vladimir I. Tishkov, Vlada B. Urlacher
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f6cb999201274ad6ae9b24f9739208e4
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spelling	oai:doaj.org-article:f6cb999201274ad6ae9b24f9739208e42021-11-08T10:55:02ZGenetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity10.1038/s41598-021-00957-52045-2322https://doaj.org/article/f6cb999201274ad6ae9b24f9739208e42021-11-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-00957-5https://doaj.org/toc/2045-2322Abstract Fusion of multiple enzymes to multifunctional constructs has been recognized as a viable strategy to improve enzymatic properties at various levels such as stability, activity and handling. In this study, the genes coding for cytochrome P450 BM3 from B. megaterium and formate dehydrogenase from Pseudomonas sp. were fused to enable both substrate oxidation catalyzed by P450 BM3 and continuous cofactor regeneration by formate dehydrogenase within one construct. The order of the genes in the fusion as well as the linkers that bridge the enzymes were varied. The resulting constructs were compared to individual enzymes regarding substrate conversion, stability and kinetic parameters to examine whether fusion led to any substantial improvements of enzymatic properties. Most noticeably, an activity increase of up to threefold was observed for the fusion constructs with various substrates which were partly attributed to the increased diflavin reductase activity of the P450 BM3. We suggest that P450 BM3 undergoes conformational changes upon fusion which resulted in altered properties, however, no NADPH channeling was detected for the fusion constructs.Arsenij KokorinPavel D. ParshinPatrick J. BakkesAnastasia A. PometunVladimir I. TishkovVlada B. UrlacherNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Arsenij Kokorin Pavel D. Parshin Patrick J. Bakkes Anastasia A. Pometun Vladimir I. Tishkov Vlada B. Urlacher Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity
description	Abstract Fusion of multiple enzymes to multifunctional constructs has been recognized as a viable strategy to improve enzymatic properties at various levels such as stability, activity and handling. In this study, the genes coding for cytochrome P450 BM3 from B. megaterium and formate dehydrogenase from Pseudomonas sp. were fused to enable both substrate oxidation catalyzed by P450 BM3 and continuous cofactor regeneration by formate dehydrogenase within one construct. The order of the genes in the fusion as well as the linkers that bridge the enzymes were varied. The resulting constructs were compared to individual enzymes regarding substrate conversion, stability and kinetic parameters to examine whether fusion led to any substantial improvements of enzymatic properties. Most noticeably, an activity increase of up to threefold was observed for the fusion constructs with various substrates which were partly attributed to the increased diflavin reductase activity of the P450 BM3. We suggest that P450 BM3 undergoes conformational changes upon fusion which resulted in altered properties, however, no NADPH channeling was detected for the fusion constructs.
format	article
author	Arsenij Kokorin Pavel D. Parshin Patrick J. Bakkes Anastasia A. Pometun Vladimir I. Tishkov Vlada B. Urlacher
author_facet	Arsenij Kokorin Pavel D. Parshin Patrick J. Bakkes Anastasia A. Pometun Vladimir I. Tishkov Vlada B. Urlacher
author_sort	Arsenij Kokorin
title	Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity
title_short	Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity
title_full	Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity
title_fullStr	Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity
title_full_unstemmed	Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity
title_sort	genetic fusion of p450 bm3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/f6cb999201274ad6ae9b24f9739208e4
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Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity

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