A Designed Supramolecular Protein Assembly with In Vivo Enzymatic Activity
The generation of new enzymatic activities has mainly relied on repurposing the interiors of preexisting protein folds because of the challenge in designing functional, three-dimensional protein structures from first principles. Here we report an artificial metallo-β-lactamase, constructed via the self-assembly of a structurally and functionally unrelated, monomeric redox protein into a tetrameric assembly that possesses catalytic zinc sites in its interfaces. The designed metallo-β-lactamase is functional in the Escherichia coli periplasm and enables the bacteria to survive treatment with ampicillin. In vivo screening of libraries has yielded a variant that displays a catalytic proficiency [(kcat/Km)/kuncat] for ampicillin hydrolysis of 2.3 × 106 and features the emergence of a highly mobile loop near the active site, a key component of natural β-lactamases to enable substrate interactions.
Importance of Scaffold Flexibility/Rigidity in the Design and Directed Evolution of Artificial Metallo-β-Lactamases
Host protein: Zn8:AB54Notes: Supramolecular protein scaffold constructed from cytochrome cb562 building blocks, Ampicillin hydrolysis: kcat/KM = 130 min-1 * M-1
Host protein: Zn8:AB54 (mutant C96T)Notes: Supramolecular protein scaffold constructed from cytochrome cb562 building blocks, Ampicillin hydrolysis: kcat/KM = 210 min-1 * M-1