6FH8

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

6FH8 の概要

• エントリーDOI: 10.2210/pdb6fh8/pdb
• 分子名称: Streptavidin, biotinylated ruthenium cyclopentadienide (3 entities in total)
• 機能のキーワード: biotin binding protein, artificial metalloenzyme, e. coli surface display, streptavidin, biotin, bioorthogonal reaction, uncaging, allyl deprotection, directed evolution
• 由来する生物種: Streptomyces avidinii
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 17215.73

構造登録者

Heinisch, T.,Schwizer, F.,Garabedian, B.,Csibra, E.,Jeschek, M.,Pinheiro Bernhardes, V.,Marliere, P.,Panke, S.,Ward, T.R. (登録日: 2018-01-12, 公開日: 2018-08-22, 最終更新日: 2024-05-08)

主引用文献

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

実験手法

X-RAY DIFFRACTION (1.64 Å)