Acyl Transfer Catalytic Activity in De Novo Designed Protein with N-Terminus of α-Helix As Oxyanion-Binding Site - Archive ouverte HAL Access content directly
Journal Articles Journal of the American Chemical Society Year : 2021

Acyl Transfer Catalytic Activity in De Novo Designed Protein with N-Terminus of α-Helix As Oxyanion-Binding Site

Abstract

Design of catalytic proteins with functional sites capable of specific chemistry gains its momentum and a number of artificial enzymes have been recently reported including hydrolases, oxidoreductases, retro-aldolases and others. Our goal is to develop a peptide ligase for robust catalysis of amide bond formation and possessing no stringent restrictions to the amino acid composition at the ligation junction. Here we report the successful completion of the first step in this long-term project by building a completely de novo protein with predefined acyl transfer catalytic activity. We applied minimalist approach to rationally design an oxyanion hole within a small cavity and containing an adjacent thiol nucleophile. The N-terminus of alpha-helix with unpaired hydrogen-bond donors was exploited as a structural motif to stabilize negatively charged tetrahedral intermediates in nucleophilic addition-elimination reactions at acyl group. Cysteine acting as a principal catalytic residue was introduced at second residue position of alpha-helix N-terminus in a designed three-alpha-helix protein based on structural informatics prediction. We showed that this minimal set of functional elements is sufficient for the emergence of catalytic activity in a de novo protein. Using peptide-thioesters as acyl-donors we demonstrated their catalyzed amidation concomitant with hydrolysis and proved that the environment at the catalytic site critically influences the reaction outcome. These results represent a promising starting point for the development of efficient catalysts for protein labeling, conjugation and peptide ligation.
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Dates and versions

hal-03196564 , version 1 (12-04-2021)

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Elise Naudin, Alastair G. Mcewen, Sophia K. Tan, Pierre Poussin-Courmontagne, Jean Louis Schmitt, et al.. Acyl Transfer Catalytic Activity in De Novo Designed Protein with N-Terminus of α-Helix As Oxyanion-Binding Site. Journal of the American Chemical Society, 2021, 143 (9), pp.3330-3339. ⟨10.1021/jacs.0c10053⟩. ⟨hal-03196564⟩
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