Publication

P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations

Beyer, N., Kulig, J. K., Bartsch, A., Hayes, M. A., Janssen, D. B. & Fraaije, M. W., Mar-2017, In : Applied Microbiology and Biotechnology. 101, p. 2319-2331 13 p.

Research output: Contribution to journalArticleAcademicpeer-review

APA

Beyer, N., Kulig, J. K., Bartsch, A., Hayes, M. A., Janssen, D. B., & Fraaije, M. W. (2017). P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations. Applied Microbiology and Biotechnology, 101, 2319-2331. https://doi.org/10.1007/s00253-016-7993-7

Author

Beyer, Nina ; Kulig, Justyna K ; Bartsch, Anette ; Hayes, Martin A ; Janssen, Dick B ; Fraaije, Marco W. / P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations. In: Applied Microbiology and Biotechnology. 2017 ; Vol. 101. pp. 2319-2331.

Harvard

Beyer, N, Kulig, JK, Bartsch, A, Hayes, MA, Janssen, DB & Fraaije, MW 2017, 'P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations', Applied Microbiology and Biotechnology, vol. 101, pp. 2319-2331. https://doi.org/10.1007/s00253-016-7993-7

Standard

P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations. / Beyer, Nina; Kulig, Justyna K; Bartsch, Anette; Hayes, Martin A; Janssen, Dick B; Fraaije, Marco W.

In: Applied Microbiology and Biotechnology, Vol. 101, 03.2017, p. 2319-2331.

Research output: Contribution to journalArticleAcademicpeer-review

Vancouver

Beyer N, Kulig JK, Bartsch A, Hayes MA, Janssen DB, Fraaije MW. P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations. Applied Microbiology and Biotechnology. 2017 Mar;101:2319-2331. https://doi.org/10.1007/s00253-016-7993-7


BibTeX

@article{e536dddfa81c4f9c817d9d16eb41728d,
title = "P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations",
abstract = "To facilitate the wider application of the NADPH-dependent P450BM3, we fused the monooxygenase with a phosphite dehydrogenase (PTDH). The resulting monooxygenase-dehydrogenase fusion enzyme acts as a self-sufficient bifunctional catalyst, accepting phosphite as a cheap electron donor for the regeneration of NADPH.The well-expressed fusion enzyme was purified and analyzed in comparison to the parent enzymes. Using lauric acid as substrate for P450BM3, it was found that the fusion enzyme had similar substrate affinity and hydroxylation selectivity while it displayed a significantly higher activity than the non-fused monooxygenase. Phosphite-driven conversions of lauric acid at restricted NADPH concentrations confirmed multiple turnovers of the cofactor. Interestingly, both the fusion enzyme and the native P450BM3 displayed enzyme concentration dependent activity and the fused enzyme reached optimal activity at a lower enzyme concentration. This suggests that the fusion enzyme has an improved tendency to form functional oligomers.To explore the constructed phosphite-driven P450BM3 as a biocatalyst, conversions of the drug compounds omeprazole and rosiglitazone were performed. PTDH-P450BM3 driven by phosphite was found to be more efficient in terms of total turnover when compared with P450BM3 driven by NADPH. The results suggest that PTDH-P450BM3 is an attractive system for use in biocatalytic and drug metabolism studies.",
keywords = "Drug metabolites, Enzyme catalysis, Fatty acids, NADPH regeneration, Oxidation, Protein engineering, BAEYER-VILLIGER MONOOXYGENASES, CYTOCHROME-P450 ENZYMES, ESCHERICHIA-COLI, FATTY-ACIDS, P450BM-3; BM3, FLAVOCYTOCHROME, HYDROXYLATION, IMMOBILIZATION, ROSIGLITAZONE",
author = "Nina Beyer and Kulig, {Justyna K} and Anette Bartsch and Hayes, {Martin A} and Janssen, {Dick B} and Fraaije, {Marco W}",
year = "2017",
month = "3",
doi = "10.1007/s00253-016-7993-7",
language = "English",
volume = "101",
pages = "2319--2331",
journal = "Applied Microbiology and Biotechnology",
issn = "0175-7598",
publisher = "SPRINGER",

}

RIS

TY - JOUR

T1 - P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations

AU - Beyer, Nina

AU - Kulig, Justyna K

AU - Bartsch, Anette

AU - Hayes, Martin A

AU - Janssen, Dick B

AU - Fraaije, Marco W

PY - 2017/3

Y1 - 2017/3

N2 - To facilitate the wider application of the NADPH-dependent P450BM3, we fused the monooxygenase with a phosphite dehydrogenase (PTDH). The resulting monooxygenase-dehydrogenase fusion enzyme acts as a self-sufficient bifunctional catalyst, accepting phosphite as a cheap electron donor for the regeneration of NADPH.The well-expressed fusion enzyme was purified and analyzed in comparison to the parent enzymes. Using lauric acid as substrate for P450BM3, it was found that the fusion enzyme had similar substrate affinity and hydroxylation selectivity while it displayed a significantly higher activity than the non-fused monooxygenase. Phosphite-driven conversions of lauric acid at restricted NADPH concentrations confirmed multiple turnovers of the cofactor. Interestingly, both the fusion enzyme and the native P450BM3 displayed enzyme concentration dependent activity and the fused enzyme reached optimal activity at a lower enzyme concentration. This suggests that the fusion enzyme has an improved tendency to form functional oligomers.To explore the constructed phosphite-driven P450BM3 as a biocatalyst, conversions of the drug compounds omeprazole and rosiglitazone were performed. PTDH-P450BM3 driven by phosphite was found to be more efficient in terms of total turnover when compared with P450BM3 driven by NADPH. The results suggest that PTDH-P450BM3 is an attractive system for use in biocatalytic and drug metabolism studies.

AB - To facilitate the wider application of the NADPH-dependent P450BM3, we fused the monooxygenase with a phosphite dehydrogenase (PTDH). The resulting monooxygenase-dehydrogenase fusion enzyme acts as a self-sufficient bifunctional catalyst, accepting phosphite as a cheap electron donor for the regeneration of NADPH.The well-expressed fusion enzyme was purified and analyzed in comparison to the parent enzymes. Using lauric acid as substrate for P450BM3, it was found that the fusion enzyme had similar substrate affinity and hydroxylation selectivity while it displayed a significantly higher activity than the non-fused monooxygenase. Phosphite-driven conversions of lauric acid at restricted NADPH concentrations confirmed multiple turnovers of the cofactor. Interestingly, both the fusion enzyme and the native P450BM3 displayed enzyme concentration dependent activity and the fused enzyme reached optimal activity at a lower enzyme concentration. This suggests that the fusion enzyme has an improved tendency to form functional oligomers.To explore the constructed phosphite-driven P450BM3 as a biocatalyst, conversions of the drug compounds omeprazole and rosiglitazone were performed. PTDH-P450BM3 driven by phosphite was found to be more efficient in terms of total turnover when compared with P450BM3 driven by NADPH. The results suggest that PTDH-P450BM3 is an attractive system for use in biocatalytic and drug metabolism studies.

KW - Drug metabolites

KW - Enzyme catalysis

KW - Fatty acids

KW - NADPH regeneration

KW - Oxidation

KW - Protein engineering

KW - BAEYER-VILLIGER MONOOXYGENASES

KW - CYTOCHROME-P450 ENZYMES

KW - ESCHERICHIA-COLI

KW - FATTY-ACIDS

KW - P450BM-3; BM3

KW - FLAVOCYTOCHROME

KW - HYDROXYLATION

KW - IMMOBILIZATION

KW - ROSIGLITAZONE

U2 - 10.1007/s00253-016-7993-7

DO - 10.1007/s00253-016-7993-7

M3 - Article

C2 - 27900443

VL - 101

SP - 2319

EP - 2331

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 0175-7598

ER -

ID: 37677965