Combinatorial reshaping of the Candida antarctica lipase A substrate pocket for enantioselectivity using an extremely condensed library
2012 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 1, 78-83 p.Article in journal (Refereed) Published
A highly combinatorial structure-based protein engineering method for obtaining enantioselectivity is reported that results in a thorough modification of the substrate binding pocket of Candida antarctica lipase A (CALA). Nine amino acid residues surrounding the entire pocket were simultaneously mutated, contributing to a reshaping of the substrate pocket to give increased enantioselectivity and activity for a sterically demanding substrate. This approach seems to be powerful for developing enantioselectivity when a complete reshaping of the active site is required. Screening toward ibuprofen ester 1, a substrate for which previously used methods had failed, gave variants with a significantly increased enantioselectivity and activity. Wild-type CALA has a moderate activity with an E value of only 3.4 toward this substrate. The best variant had an E value of 100 and it also displayed a high activity. The variation at each mutated position was highly reduced, comprising only the wild type and an alternative residue, preferably a smaller one with similar properties. These minimal binary variations allow for an extremely condensed protein library. With this highly combinatorial method synergistic effects are accounted for and the protein fitness landscape is explored efficiently.
Place, publisher, year, edition, pages
2012. Vol. 109, no 1, 78-83 p.
kinetic resolution, library design, protein design, enzyme catalysis
Research subject Organic Chemistry
IdentifiersURN: urn:nbn:se:su:diva-74997DOI: 10.1073/pnas.1111537108ISI: 000298876500022OAI: oai:DiVA.org:su-74997DiVA: diva2:513486
FunderSwedish Research Council, 621-2010-4737Knut and Alice Wallenberg FoundationSwedish Foundation for Strategic Research