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Modular assembly of phosphite dehydrogenase and phenylacetone monooxygenase for tuning cofactor regeneration
- Source :
- Biomolecules, Volume 11, Issue 6, Biomolecules, Vol 11, Iss 905, p 905 (2021), Biomolecules, 11(6):905. MDPI AG
- Publication Year :
- 2021
- Publisher :
- MDPI AG, 2021.
-
Abstract
- The use of multienzyme complexes can facilitate biocatalytic cascade reactions by employing fusion enzymes or protein tags. In this study, we explored the use of recently developed peptide tags that promote complex formation of the targeted proteins: the dimerization-docking and anchoring domain (RIDD–RIAD) system. These peptides allow self-assembly based on specific protein–protein interactions between both peptides and allow tuning of the ratio of the targeted enzymes as the RIAD peptide binds to two RIDD peptides. Each of these tags were added to the C-terminus of a NADPH-dependent Baeyer–Villiger monooxygenase (phenylacetone monooxygenase, PAMO) and a NADPH-regenerating enzyme (phosphite dehydrogenase, PTDH). Several RIDD/RIAD-tagged PAMO and PTDH variants were successfully overproduced in E. coli and subsequently purified. Complementary tagged enzymes were mixed and analyzed for their oligomeric state, stability, and activity. Complexes were formed in the case of some specific combinations (PAMORIAD–PTDHRIDD and PAMORIAD/RIAD–PTDHRIDD). These enzyme complexes displayed similar catalytic activity when compared with the PTDH–PAMO fusion enzyme. The thermostability of PAMO in these complexes was retained while PTDH displayed somewhat lower thermostability. Evaluation of the biocatalytic performance by conducting conversions revealed that with a self-assembled PAMO–PTDH complex less PTDH was required for the same performance when compared with the PTDH–PAMO fusion enzyme.
- Subjects :
- 0301 basic medicine
RIAD–RIDD tag
Peptide
Protein tag
Baeyer–Villiger monooxygenase
01 natural sciences
Biochemistry
Microbiology
Cofactor
Article
Catalysis
Mixed Function Oxygenases
03 medical and health sciences
Multienzyme Complexes
NADH, NADPH Oxidoreductases
Molecular Biology
Phenylacetone monooxygenase
Thermostability
chemistry.chemical_classification
biology
010405 organic chemistry
Chemistry
Cofactor regeneration
Self-assembly
Monooxygenase
Recombinant Proteins
QR1-502
0104 chemical sciences
030104 developmental biology
Enzyme
Oligomers
biology.protein
Subjects
Details
- Language :
- English
- ISSN :
- 2218273X
- Volume :
- 11
- Issue :
- 6
- Database :
- OpenAIRE
- Journal :
- Biomolecules
- Accession number :
- edsair.doi.dedup.....d8a79074d6ef386e9ca5cf302b2376c1