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New recombinant cyclohexylamine oxidase variants for deracemization of secondary amines by orthogonally assaying designed mutants with structurally diverse substrates.
- Source :
-
Scientific reports [Sci Rep] 2016 May 03; Vol. 6, pp. 24973. Date of Electronic Publication: 2016 May 03. - Publication Year :
- 2016
-
Abstract
- To further expand the substrate range of the cyclohexylamine oxidase (CHAO) from Brevibacterium oxydans, a library of diverse mutants was created and assayed toward a group of structurally diverse substrates. Among them, mutants T198A and M226A exhibited enhanced activity relative to wt CHAO for most (S)-enantiomers of primary amines and some secondary amines. While mutants T198I, L199I, L199F, M226I and M226T were more active than wt CHAO toward the primary amines, mutants T198F, L199T, Y321A, Y321T, Y321I and Y321F enhanced the enzyme activity toward the secondary amines. In particular, mutant Y321I displayed an enhanced catalytic efficiency toward 1-(4-methoxybenzyl)-1, 2, 3, 4, 5, 6, 7, 8-octahydroisoquinoline (13). Whereas a double mutant, Y321I/M226T, acted on (S)-N-(prop-2-yn-1-yl)-2, 3-dihydro-1H-inden-1-amine [(S)-8]. Since (R)-8 is an irreversible inhibitor of monoamine oxidase and (S)-13 is an intermediate of dextromethorphan, a cough suppressant drug, deracemizations of 8 and 13 were carried out with crude enzyme extracts of the respective mutants. This resulted in 51% and 78% isolated yields of (R)-8 and (S)-13, respectively, each with high enantiomeric excess (93% and 99% ee). The results demonstrated the application potential of the evolved CHAO mutants in drug synthesis requiring chiral secondary amines.
- Subjects :
- Actinobacteria genetics
Isomerism
Mutant Proteins genetics
Mutant Proteins metabolism
Mutation, Missense
Oxidoreductases Acting on CH-NH Group Donors genetics
Recombinant Proteins genetics
Substrate Specificity
Actinobacteria enzymology
Amines metabolism
Oxidoreductases Acting on CH-NH Group Donors metabolism
Recombinant Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 2045-2322
- Volume :
- 6
- Database :
- MEDLINE
- Journal :
- Scientific reports
- Publication Type :
- Academic Journal
- Accession number :
- 27138090
- Full Text :
- https://doi.org/10.1038/srep24973