A designed supramolecular protein assembly with in vivo enzymatic activity

Abstract

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 [(k(cat)/K(m))/k(uncat)] for ampicillin hydrolysis of 2.3 × 10(6) and features the emergence of a highly mobile loop near the active site, a key component of natural β-lactamases to enable substrate interactions.

A Photoactive Carbon‐Monoxide‐Releasing Protein Cage for Dose‐Regulated Delivery in Living Cells

“…[8][9][10] Protein assemblies containing metal compounds are biocompatible and have been recently used for both in vitro and in vivo applications. [11][12][13] Metal-based drugs,i maging reagents,a nd metal nanoparticles can be encapsulated within protein assemblies,a nd delivered into living cells since the proteins retain their coordination structures within the in vivo environment. [13][14][15] Although several stimulus-responsive composites have been investigated in biological applications at the nanoscale,there have been few reports of functionalizing the composites in living cells.…”

Installing hydrolytic activity into a completely de novo protein framework

“…[20][21][22] Although more challenging, this would carry advantages over redesign approaches: firstly, the designer could select and control all, or at least many of the residues, and so engineer the complete construct predictably; secondly, the resulting proteins could be made to function under conditions away from those required by natural proteins; and finally, success in this area would provide the acid test of our understanding of enzyme structure and function. Towards this fully de novo effort, successful designs of hydrolases have included: the decoration of small protein-folding motifs with His residues; 23,24 the employment of Zn 2+ cations as Lewisacidic cofactors; [25][26][27][28] and the identification of catalytically active proteins in combinatorial libraries of sequences patterned to form to all- or all- protein folds. 29 One of the most productive areas in the de novo design of protein structure has been for coiled-coil assemblies.…”

Genetic Optimization of Metalloenzymes: Enhancing Enzymes for Non‐Natural Reactions

“…[65] In the course of these studies,m etal binding was used to create and maintain the quaternary structure,w hile other metal centers served as catalysts.Appreciable activity was observed on p-nitrophenylacetate and ampicilline when af lanking lysine residues was mutated to alanine in the artificial folds generated from zinc and cb562 (a heme-containing four-helix bundle). Them utation of residues proximal to the catalytically active zinc-binding site provided amutant with superior activity.…”