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Modular Assembly of Phosphite Dehydrogenase and Phenylacetone Monooxygenase for Tuning Cofactor Regeneration
- Source :
- Biomolecules, Vol 11, Iss 6, p 905 (2021)
- 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.
Details
- Language :
- English
- ISSN :
- 11060905 and 2218273X
- Volume :
- 11
- Issue :
- 6
- Database :
- Directory of Open Access Journals
- Journal :
- Biomolecules
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.9abf52ae265437b8d26a00cf48da9a8
- Document Type :
- article
- Full Text :
- https://doi.org/10.3390/biom11060905