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

Cited 37 time in Web of Science Cited 38 time in Scopus
Authors

Song, Woon Ju; Yu, Jaeseung; Tezcan, F. Akif

Issue Date
2017-11
Publisher
American Chemical Society
Citation
Journal of the American Chemical Society, Vol.139 No.46, pp.16772-16779
Abstract
We describe the design and evolution of catalytic hydrolase activity on a supramolecular protein scaffold, Zn-4:(C96)RIDC1(4), which was constructed from cytochrome cb(s62) building blocks via a metal-templating strategy. Previously, we reported that Zn4:(C96)RIDC1(4) could be tailored with tripodal (His/His/Glu), unsaturated Zn coordination motifs in its interfaces to generate a variant termed Zn-8,(A104)AB3(4), which in turn displayed catalytic activity for the hydrolysis of activated esters and beta-lactam antibiotics. zn(8):(A104)AB3(4) was subsequently subjected to directed evolution via an in vivo selection strategy, leading to a variant Zn-8:(A104/G57)AB3(4) which displayed enzyme-like Michaelis-Menten behavior for ampicillin hydrolysis. A criterion for the evolutionary utility or designability of a new protein structure is its ability to accommodate different active sites. With this in mind, we examined whether Zn-4:(C96)RIDC1(4) could be tailored with alternative Zn coordination sites that could similarly display evolvable catalytic activities. We report here a detailed structural and functional characterization of new variant Zn-8:AB5(4), which houses similar, unsaturated Zn coordination sites to those in Zn-8:(A104/G57)AB3(4)) but in completely different microenvironments. Zn-8:AB5(4) displays Michaelis Menten behavior for ampicillin hydrolysis without any optimization. Yet, the subsequent directed evolution of Zn-8:AB5(4) revealed limited catalytic improvement, which we ascribed to the local protein rigidity surrounding the Zn centers and the lack of evolvable loop structures nearby. The relaxation of local rigidity via the elimination of adjacent disulfide linkages led to a considerable structural transformation with a concomitant improvement in beta-lactamase activity. Our findings reaffirm previous observations that the delicate balance between protein flexibility and stability is crucial for enzyme design and evolution.
ISSN
0002-7863
URI
https://hdl.handle.net/10371/202939
DOI
https://doi.org/10.1021/jacs.7b08981
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  • College of Natural Sciences
  • Department of Chemistry
Research Area Biochemistry, Inorganic, 무기화학, 생화학

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