Reversed Enantiopreference of an omega-Transaminase by a Single-Point Mutation
2010 (English)In: CHEMCATCHEM, ISSN 1867-3880, Vol. 2, no 8, 976-980 p.Article in journal (Refereed) Published
Altering the characteristics of an active-site loop in an (S)-selective omega-transaminase from Arthrobacter citreus (variant CNB05-01) influences the enantioselectivity. This active-site loop belongs to the second subunit of the dimeric enzyme structure that participates in the coordination of pyridoxal-5'-phosphate (PLP) in the so called "phosphate group binding cup". Three amino acid residues (E326, V328, and Y331) in this loop are selected by homology modeling for site-directed mutagenesis aiming to increase the enzyme enantioselectivity for 4-fluorophenylacetone. By combining these mutations, five enzyme variants are created. The performance of these variants is explored using a model system consisting of isopropylamine and 4-fluorophenylacetone or 4-nitroacetophenone in asymmetric synthesis using a whole-cell system approach. Three of the five variants show increased enantioselectivity for 4-fluorophenylacetone compared to CNB05-01. Variant CNB05-01/Y331C increases the enantioselectivity from 98% ee to over 99.5% ee. A single-point mutation, V328A, turn the (S)-selective omega-transaminase into an (R)-selective enzyme. This switch in enantioselectivity is substrate dependent, exhibiting (R) selectivity for 4-fluorophenylacetone and retaining (S) selectivity for 4-nitroacetophenone. The shift in enantiopreference is further confirmed by molecular docking simulations. Homology modeling is shown to be a powerful tool to target important amino acid residues in this enzyme in order to improve enantioselectivity by rational design.
Place, publisher, year, edition, pages
2010. Vol. 2, no 8, 976-980 p.
amines, asymmetric synthesis, enantioselectivity, enzymes, rational design
Biochemistry and Molecular Biology
IdentifiersURN: urn:nbn:se:kth:diva-26834DOI: 10.1002/cctc.201000107ISI: 000281321700013ScopusID: 2-s2.0-79957694192OAI: oai:DiVA.org:kth-26834DiVA: diva2:374790
QC 201012062010-12-062010-11-292010-12-06Bibliographically approved