Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity.
Bacillus megaterium
/ enzymology
Bacterial Proteins
/ genetics
Catalysis
Cytochrome P-450 Enzyme System
/ genetics
Enzymes
/ genetics
Formate Dehydrogenases
/ genetics
NADP
/ metabolism
NADPH-Ferrihemoprotein Reductase
/ genetics
Oxidation-Reduction
Protein Engineering
/ methods
Pseudomonas
/ enzymology
Recombinant Fusion Proteins
/ genetics
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
04 11 2021
04 11 2021
Historique:
received:
12
08
2021
accepted:
08
10
2021
entrez:
5
11
2021
pubmed:
6
11
2021
medline:
27
1
2022
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 34737365
doi: 10.1038/s41598-021-00957-5
pii: 10.1038/s41598-021-00957-5
pmc: PMC8568981
doi:
Substances chimiques
Bacterial Proteins
0
Enzymes
0
Recombinant Fusion Proteins
0
NADP
53-59-8
Cytochrome P-450 Enzyme System
9035-51-2
Formate Dehydrogenases
EC 1.17.1.9
NADPH-Ferrihemoprotein Reductase
EC 1.6.2.4
flavocytochrome P450 BM3 monoxygenases
EC 1.6.2.4
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
21706Subventions
Organisme : European Regional Development Fund
ID : 34.EFRE-0300095/1703FI04
Organisme : Russian Science Foundation
ID : 18-74-0014
Informations de copyright
© 2021. The Author(s).
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