6FH8

E. coli surface display of streptavidin for directed evolution of an allylic deallocase

Summary for 6FH8

• Entry DOI: 10.2210/pdb6fh8/pdb
• Descriptor: Streptavidin, biotinylated ruthenium cyclopentadienide (3 entities in total)
• Functional Keywords: biotin binding protein, artificial metalloenzyme, e. coli surface display, streptavidin, biotin, bioorthogonal reaction, uncaging, allyl deprotection, directed evolution
• Biological source: Streptomyces avidinii
• Total number of polymer chains: 1
• Total formula weight: 17215.73

Authors

Heinisch, T.,Schwizer, F.,Garabedian, B.,Csibra, E.,Jeschek, M.,Pinheiro Bernhardes, V.,Marliere, P.,Panke, S.,Ward, T.R. (deposition date: 2018-01-12, release date: 2018-08-22, Last modification date: 2024-05-08)

Primary citation

Heinisch, T.,Schwizer, F.,Garabedian, B.,Csibra, E.,Jeschek, M.,Vallapurackal, J.,Pinheiro, V.B.,Marliere, P.,Panke, S.,Ward, T.R.
E. colisurface display of streptavidin for directed evolution of an allylic deallylase.
Chem Sci, 9:5383-5388, 2018

PubMed 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 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 aminocoumarin . Two rounds of directed evolution afforded the double mutant S112M-K121A that displayed a 36-fold increase in surface activity cellular background and a 5.7-fold increased 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.

PubMed: 30079176
DOI: 10.1039/c8sc00484f

Experimental method

X-RAY DIFFRACTION (1.64 Å)