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Installing hydrolytic activity into a completely de novo protein framework

DOI: 10.1038/nchem.2555 DOI Help

Authors: Antony J. Burton (University of Bristol) , Andrew R. Thomson (University of Bristol) , William M. Dawson (University of Bristol) , R. Leo Brady (University of Bristol) , Derek N. Woolfson (University of Bristol; BrisSynBio)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nature Chemistry , VOL 8 , PAGES 837 - 844

State: Published (Approved)
Published: July 2016
Diamond Proposal Number(s): 8922

Abstract: The design of enzyme-like catalysts tests our understanding of sequence-to-structure/function relationships in proteins. Here we install hydrolytic activity predictably into a completely de novo and thermostable α-helical barrel, which comprises seven helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine–histidine–glutamic acid triads on each helix, hydrolyses p-nitrophenyl acetate with catalytic efficiencies that match the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis.

Journal Keywords: Biocatalysis; Hydrolases; Protein design

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I03-Macromolecular Crystallography , I04-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography

Added On: 17/11/2016 15:20

Discipline Tags:

Biochemistry Catalysis Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Macromolecular Crystallography (MX)