E. coli surface display of streptavidin for directed evolution of an allylic deallylase. Issue 24 (31st May 2018)

• Record Type: Journal Article
• Authors: Heinisch, Tillmann; Schwizer, Fabian; Garabedian, Brett; Csibra, Eszter; Jeschek, Markus; Vallapurackal, Jaicy; Pinheiro, Vitor B.; Marlière, Philippe; Panke, Sven; Ward, Thomas R.
• Is Part Of: Chemical science. Volume 9:Issue 24(2018)
• Page Start: 5383
• Page End: 5388
• Publication Date: 2018-05-31
• Subjects: Chemistry -- Periodicals; 540.5
• DOI: 10.1039/c8sc00484f
• Languages: English
• ISSNs: 2041-6520

Abstract

Abstract : An artificial deallylase is constituted on the E. coli surface and genetically optimized for the deprotection of caged aminocoumarin. Abstract : Artificial metalloenzymes (ArMs hereafter) combine attractive features of both homogeneous catalysts and enzymes and offer the potential to implement new-to-nature reactions in living organisms. Herein we present an E. coli surface display platform for streptavidin (Sav hereafter) relying on an Lpp-OmpA anchor. The system was used for the high throughput screening of a bioorthogonal CpRu-based artificial deallylase (ADAse) that uncages an allylcarbamate-protected aminocoumarin1 . Two rounds of directed evolution afforded the double mutant S112M–K121A that displayed a 36-fold increase in surface activity vs. cellular background and a 5.7-fold increased in vitro activity compared to the wild type enzyme. The crystal structure of the best ADAse reveals the importance of mutation S112M to stabilize the cofactor conformation inside the protein.