Combinatorial reshaping of the Candida antarctica lipase A substrate pocket for enantioselectivity using an extremely condensed library

• Published in: Proceedings of the National Academy of Sciences - PNAS 2012-01, Vol.109 (1), p.78-83
• Main Authors: Sandström, Anders G., Wikmark, Ylva, Engström, Karin, Nyhlén, Jonas, Bäckvall, Jan-E.
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Amino acids; Antarctic regions; Binding sites; Biological Sciences; Candida - enzymology; Candida antarctica; Catalytic Domain; Directed molecular evolution; Ecological competition; enzyme catalysis; Enzyme kinetics; Enzyme substrates; Enzymes; Esters; Esters - chemistry; Homologous Recombination - genetics; Ibuprofen - analysis; Ibuprofen - chemistry; kinetic resolution; Libraries; library design; Lipase - chemistry; Lipase - metabolism; Models, Molecular; Mutagenesis; Mutagenesis - genetics; Mutant Proteins - chemistry; Mutation; Mutation - genetics; Organic Chemistry; organisk kemi; Peptide Library; Physical Sciences; Protein Conformation; protein design; Protein engineering; Proteins; Stereoisomerism; Substrate Specificity
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1111537108

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.