title: Installing hydrolytic activity into a completely de novo protein framework.
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.
authors.affiliation: School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, UK.
authors.forename: Antony J
authors.initials: AJ
authors.lastname: Burton
authors.affiliation: School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, UK.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Thomson
authors.affiliation: School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, UK.
authors.forename: William M
authors.initials: WM
authors.lastname: Dawson
authors.affiliation: School of Biochemistry, University of Bristol, Medical Sciences Building, University Walk, Bristol BS8 1TD, UK.
authors.forename: R Leo
authors.initials: RL
authors.lastname: Brady
authors.affiliation: School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, UK.|School of Biochemistry, University of Bristol, Medical Sciences Building, University Walk, Bristol BS8 1TD, UK.|BrisSynBio, Life Sciences Building, Tyndall Avenue, Bristol BS8 1TQ, UK.
authors.forename: Derek N
authors.identifier: 0000-0002-0394-3202
authors.initials: DN
authors.lastname: Woolfson
mesh_terms: D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D009596:Nitrophenols / Q000737:chemistry; D000072756:Protein Conformation, alpha-Helical; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary
chemical_list: D009596:Nitrophenols; C008642:4-nitrophenyl acetate; D002265:Carboxylic Ester Hydrolases