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Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

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αRep A3: A Versatile Artificial Scaffold for Metalloenzyme Design.

Thibault Di Meo^1,2, 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.

Keywords:

Diels-Alder reaction artificial enzymes enantioselective catalysis green chemistry protein engineering

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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.