Cargando…

P450(BM3) fused to phosphite dehydrogenase allows phosphite-driven selective oxidations

To facilitate the wider application of the NADPH-dependent P450(BM3,) 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 regen...

Descripción completa

Detalles Bibliográficos
Autores principales: Beyer, Nina, Kulig, Justyna K., Bartsch, Anette, Hayes, Martin A., Janssen, Dick B., Fraaije, Marco W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Berlin Heidelberg 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320008/
https://www.ncbi.nlm.nih.gov/pubmed/27900443
http://dx.doi.org/10.1007/s00253-016-7993-7
Descripción
Sumario:To facilitate the wider application of the NADPH-dependent P450(BM3,) 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 P450(BM3), 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 P450(BM3) 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 P450(BM3) as a biocatalyst, conversions of the drug compounds omeprazole and rosiglitazone were performed. PTDH-P450(BM3) driven by phosphite was found to be more efficient in terms of total turnover when compared with P450(BM3) driven by NADPH. The results suggest that PTDH-P450(BM3) is an attractive system for use in biocatalytic and drug metabolism studies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00253-016-7993-7) contains supplementary material, which is available to authorized users.