Catalytic performance of a class III old yellow enzyme and its cysteine variants
- Class III old yellow enzymes (OYEs) contain a conserved cysteine in their active sites. To address the role of this cysteine in OYE-mediated asymmetric synthesis, we have studied the biocatalytic properties of OYERo2a from \(\textit {Rhodococcus opacus}\) 1CP (WT) as well as its engineered variants C25A, C25S and C25G. OYERo2a in its redox resting state (oxidized form) is irreversibly inactivated by N-methylmaleimide. As anticipated, inactivation does not occur with the Cys variants. Steady-state kinetics with this maleimide substrate revealed that C25S and C25G doubled the turnover frequency (\(\it {k}\)\(_{cat}\)) while showing increased K\(_{M}\) values compared to WT, and that C25A performed more similar to WT. Applying the substrate 2-cyclohexen-1-one, the Cys variants were less active and less efficient than WT. OYERo2a and its Cys variants showed different activities with NADPH, the natural reductant. The variants did bind NADPH less well but \(\it {k}\)\(_{cat}\) was significantly increased. The most efficient variant was C25G. Replacement of NADPH with the cost-effective synthetic cofactor 1-benzyl-1,4-dihydronicotinamide (BNAH) drastically changed the catalytic behavior. Again C25G was most active and showed a similar efficiency as WT. Biocatalysis experiments showed that OYERo2a, C25S, and C25G converted \(\it{N}\)-phenyl-2-methylmaleimide equally well (81–84%) with an enantiomeric excess (\(\it{ee}\)) of more than 99% for the R-product. With cyclic ketones, the highest conversion (89%) and (\it{ee}\) (>99%) was observed for the reaction of WT with \(\it{R}\)-carvone. A remarkable poor conversion of cyclic ketones occurred with C25G. In summary, we established that the generation of a cysteine-free enzyme and cofactor optimization allows the development of more robust class III OYEs.
Author: | Anika ScholtissekGND, Eric GädkeGND, Caroline E. PaulORCiDGND, Arie H. WestphalGND, Willem J. H. van BerkelORCiDGND, Dirk TischlerORCiDGND |
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URN: | urn:nbn:de:hbz:294-64085 |
DOI: | https://doi.org/10.3389/fmicb.2018.02410 |
Parent Title (English): | Frontiers in microbiology |
Document Type: | Article |
Language: | English |
Date of Publication (online): | 2019/05/03 |
Date of first Publication: | 2018/10/12 |
Publishing Institution: | Ruhr-Universität Bochum, Universitätsbibliothek |
Tag: | Open Access Fonds Rhodococcus opacus 1CP; actinobacteria; biocatalysis; cysteine modification; ene reductase; flavoprotein; inactivation; protein engineering |
Volume: | 9 |
First Page: | 2410-1 |
Last Page: | 2410-10 |
Note: | Frontiers in microbiology, Bd. 9, Artikelnummer 2410 |
Note: | Article Processing Charge funded by the Deutsche Forschungsgemeinschaft (DFG) and the Open Access Publication Fund of Ruhr-Universität Bochum. |
Institutes/Facilities: | Lehrstuhl für Pflanzenphysiologie, Nachwuchsgruppe Mikrobielle Biotechnologie |
Dewey Decimal Classification: | Naturwissenschaften und Mathematik / Biowissenschaften, Biologie, Biochemie |
open_access (DINI-Set): | open_access |
faculties: | Fakultät für Biologie und Biotechnologie |
Licence (English): | Creative Commons - CC BY-NC 4.0 - Attribution-NonCommercial 4.0 International |