Installing hydrolytic activity into a completely de novo protein framework

Burton, A. J., Thomson, A. R. , Dawson, W. M., Brady, R. L. and Woolfson, D. N. (2016) Installing hydrolytic activity into a completely de novo protein framework. Nature Chemistry, 8(9), pp. 837-844. (doi: 10.1038/nchem.2555) (PMID:27554410)

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.

• Item Type: Articles
• Additional Information: A.J.B. thanks the Bristol Chemical Synthesis Centre for Doctoral Training funded by the Engineering and Physical Sciences Research Council (EP/G036764/1) and the University of Bristol for the provision of a PhD studentship. A.J.B., A.R.T., W.M.D. and D.N.W. are supported by the European Research Council (340764). D.N.W. holds a Royal Society Wolfson Research Merit Award.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Thomson, Dr Drew
• Authors: Burton, A. J., Thomson, A. R., Dawson, W. M., Brady, R. L., and Woolfson, D. N.
• College/School: College of Science and Engineering > School of Chemistry
• Journal Name: Nature Chemistry
• Publisher: Nature Publishing Group
• ISSN: 1755-4330
• ISSN (Online): 1755-4349
• Published Online: 04 July 2016
• Copyright Holders: Copyright © 2016 Macmillan Publishers Limited, part of Springer Nature
• First Published: First published in Nature Chemistry 8(9): 837-844
• Publisher Policy: Reproduced in accordance with the publisher copyright policy