Functionalized Artificial Bidomain Proteins Based on an α-Solenoid Protein Repeat Scaffold: A New Class of Artificial Diels–Alderases

Abstract

αRep is a family of entirely artificial repeat proteins. Within the previously described αRep library, some variants are homodimers displaying interdomain cavities. Taking advantage of these properties, one of these homodimers called αRep A3 was converted into entirely artificial single chain bidomain metalloenzymes. A nonmutated A3 domain was covalently linked with an A3′ domain bearing a unique cysteine on a chosen mutated position (F119C or Y26C). This single mutation ensured the covalent coupling of a 1:1 copper(II)/phenanthroline or copper(II)/terpyridine complex as a catalytic center within the interdomain cavity which was maintained large enough to accommodate two substrates of the Diels–Alder (D–A) reaction. This allowed us to obtain four new artificial Diels–Alderases that were fully characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, UV–vis spectroscopy, and size exclusion chromatography analyses and were then further used for the catalysis of the D–A r...