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Publication Abstract from PubMed

The design of catalytic proteins with functional sites capable of specific chemistry is gaining momentum and a number of artificial enzymes have recently been reported, including hydrolases, oxidoreductases, retro-aldolases, and others. Our goal is to develop a peptide ligase for robust catalysis of amide bond formation that possesses no stringent restrictions to the amino acid composition at the ligation junction. We report here 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 a minimalist approach to rationally design an oxyanion hole within a small cavity that contains an adjacent thiol nucleophile. The N-terminus of the 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 the acyl group. Cysteine acting as a principal catalytic residue was introduced at the second residue position of the 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-(alpha)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.

Acyl Transfer Catalytic Activity in De Novo Designed Protein with N-Terminus of alpha-Helix As Oxyanion-Binding Site.,Naudin EA, McEwen AG, Tan SK, Poussin-Courmontagne P, Schmitt JL, Birck C, DeGrado WF, Torbeev V J Am Chem Soc. 2021 Mar 10;143(9):3330-3339. doi: 10.1021/jacs.0c10053. Epub 2021 , Feb 26. PMID:33635059^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.