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 aminocoumarin 1. 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.
Highlights
A new generation of green, sustainable and biocompatible catalysts is a prerequisite to produce the ne chemicals and complex drugs of the future.[1]
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
As a proofof-principle, we demonstrated that a Sav-based arti cial metalloenzyme for ring-closing metathesis could be assembled and evolved in the periplasm of E. coli.[19]
Summary
A new generation of green, sustainable and biocompatible catalysts is a prerequisite to produce the ne chemicals and complex drugs of the future.[1]. Similar effects have been reported in other Lpp-OmpA-labeling studies.[30] Fluorescence microscopy revealed E. coli uorescence labeling only in the presence of cells with Sav displayed on the surface. The ADAse [CpRu(QA-Biot)(OH2)] 3$WT Sav had a 1.5-fold increase over cellular background (Fig. S18†).
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