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A designed supramolecular protein assembly with in vivo enzymatic activity.

Science (New York, N.Y.) (2014-12-20)
Woon Ju Song, F Akif Tezcan
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Zinc, sputtering target, diam. × thickness 3.00 in. × 0.125 in., 99.995% trace metals basis
Zinc, foil, 4mm disks, thickness 0.175mm, as rolled, 99.95+%
Zinc, foil, 25mm disks, thickness 0.5mm, as rolled, 99.95+%
Zinc, foil, 2m coil, thickness 0.35mm, as rolled, 99.95+%
Zinc, foil, 5m coil, thickness 0.025mm, as rolled, 99.95+%
Zinc, foil, 4mm disks, thickness 0.5mm, as rolled, 99.99+%
Zinc, foil, 2m coil, thickness 0.1mm, as rolled, 99.95+%
Zinc, foil, 50mm disks, thickness 0.5mm, as rolled, 99.99+%
Zinc, foil, 4mm disks, thickness 0.05mm, as rolled, 99.95+%
Zinc, foil, 4mm disks, thickness 0.25mm, as rolled, 99.99+%
Zinc, foil, 2m coil, thickness 0.025mm, as rolled, 99.95+%
Zinc, foil, 5m coil, thickness 0.05mm, as rolled, 99.95+%
Zinc, foil, 25mm disks, thickness 0.5mm, as rolled, 99.99+%
Zinc, foil, 50mm disks, thickness 1.0mm, as rolled, 99.95+%
Zinc, foil, 4mm disks, thickness 0.05mm, as rolled, 99.99+%
Zinc, foil, 2m coil, thickness 0.05mm, as rolled, 99.95+%
Zinc, foil, 6mm disks, thickness 0.5mm, as rolled, 99.99+%
Zinc, foil, 8mm disks, thickness 1.0mm, as rolled, 99.95+%
Zinc, foil, 6mm disks, thickness 0.125mm, as rolled, 99.99+%
Zinc, foil, light tested, 25x25mm, thickness 0.025mm, as rolled, 99.99+%
Zinc, foil, 8mm disks, thickness 0.15mm, as rolled, 99.95+%
Zinc, foil, 2m coil, thickness 0.5mm, as rolled, 99.95+%
Sigma-Aldrich
Zinc, shot, 99.9999%
Supelco
Ampicillin trihydrate, VETRANAL®, analytical standard
Sigma-Aldrich
Ampicillin, meets USP testing specifications
Sigma-Aldrich
Ampicillin trihydrate, 900-1050 μg/mg anhydrous basis (HPLC)
Sigma-Aldrich
Zinc, granular, 20-30 mesh, ACS reagent, ≥99.8%
Sigma-Aldrich
Zinc, shot, 5 mm, 99.999% trace metals basis
Sigma-Aldrich
Zinc, foil, thickness 0.25 mm, 99.999% trace metals basis
Sigma-Aldrich
Zinc, foil, thickness 1.0 mm, 99.99% trace metals basis