Importance of Scaffold Flexibility/Rigidity in the Design and Directed Evolution of Artificial Metallo-β-lactamases

• DOI: 10.1021/jacs.7b08981
• Publication Date: 2017-10-10T06:08:38Z
• Authors: Woon Ju Song; Jaeseung Yu; F. Akif Tezcan
• Keywords: Rigidity (electromagnetism)

We describe the design and evolution of catalytic hydrolase activity on a supramolecular protein scaffold, Zn4:C96RIDC14, which was constructed from cytochrome cb562 building blocks via metal-templating strategy. Previously, we reported that Zn4:C96RIDC14 could be tailored with tripodal (His/His/Glu), unsaturated Zn coordination motifs in its interfaces to generate variant termed Zn8:A104AB34, turn displayed for hydrolysis activated esters β-lactam antibiotics. Zn8:A104AB34 subsequently subjected directed an vivo selection strategy, leading Zn8:A104/G57AB34 enzyme-like Michaelis-Menten behavior ampicillin hydrolysis. A criterion evolutionary utility or designability new structure is ability accommodate different active sites. With this mind, examined whether alternative sites similarly display evolvable activities. report here detailed structural functional characterization Zn8:AB54, houses similar, those Zn8:A104/G57AB34, but completely microenvironments. Zn8:AB54 displays without any optimization. Yet, subsequent revealed limited improvement, ascribed local rigidity surrounding centers lack loop structures nearby. The relaxation elimination adjacent disulfide linkages led considerable transformation concomitant improvement β-lactamase activity. Our findings reaffirm previous observations delicate balance between flexibility stability crucial enzyme evolution.