title: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0356, USA.
authors.forename: Woon Ju
authors.initials: WJ
authors.lastname: Song
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0356, USA. tezcan@ucsd.edu.
authors.forename: F Akif
authors.initials: FA
authors.lastname: Tezcan
mesh_terms: D000667:Ampicillin / Q000737:chemistry* / Q000494:pharmacology; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology; D006868:Hydrolysis; D008667:Metalloproteins / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D019897:Periplasm / Q000201:enzymology; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry*; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics
chemical_list: D008667:Metalloproteins; D000667:Ampicillin; D001618:beta-Lactamases; D015032:Zinc