αRep A3: A Versatile Artificial Scaffold for Metalloenzyme Design
Thibault Di Meo1,2, Wadih Ghattas1, Christian Herrero1
1Institut de Chimie Moléculaire et des Matériaux d'Orsay (ICMMO), UMR 8182, CNRS, Univ. Paris Sud, Université Paris-Saclay, Bât. 420, rue du Doyen Georges Poitou, 91405, Orsay cedex, France.
Chemistry (Weinheim an Der Bergstrasse, Germany)
|May 26, 2017
Summary
Artificial proteins called αRep A3 were engineered into novel biocatalysts. These biohybrids demonstrated selective copper binding and catalyzed enantioselective Diels-Alder reactions, showcasing a new route for artificial enzyme design.
Area of Science:
- Protein Engineering
- Artificial Biocatalysis
- Synthetic Biology
Background:
- αRep proteins are a novel class of artificial proteins built on a thermostable α-helical motif.
- The αRep A3 dimer possesses a unique cleft suitable for accommodating metal complexes, suggesting potential for biocatalyst development.
Purpose of the Study:
- To engineer the αRep A3 protein scaffold for creating novel artificial biocatalysts.
- To covalently attach a phenanthroline ligand to specific cysteine residues in αRep A3 variants.
- To investigate the metal-binding capabilities and catalytic activity of the resulting biohybrids.
Main Methods:
- Site-directed mutagenesis was used to introduce cysteine residues at positions F119 and Y26 of the αRep A3 dimer.
- A phenanthroline ligand was covalently attached to the engineered cysteine residues.
- Purification, characterization, copper(II) binding studies, and enantioselective Diels-Alder cycloaddition assays were performed.
Main Results:
- Mutated and ligand-coupled αRep A3 variants retained their folded, dimeric structure.
- The biohybrids specifically bound copper(II) ions through distinct coordination modes.
- The holo-biohybrid A3F119NPH achieved up to 62% enantiomeric excess in catalyzing Diels-Alder reactions.
Conclusions:
- The αRep A3 dimer serves as a validated and effective scaffold for designing artificial biohybrids.
- This study presents a promising strategy for the development of enantioselective artificial biocatalysts derived from artificial proteins.
