Artificial metalloenzymes enabled by combining proteins with hemin via protein refolding

Abstract

In this study, we unveil a conceptual technology for fabricating artificial metalloenzymes (ArMs) by deeply integrating hemin into protein scaffolds via a protein refolding process, a method that transcends the conventional scope of surface-level modifications. Our approach involves denaturing proteins, such as benzaldehyde lyase, green fluorescent protein, and Candida antarctica lipase B, to expose extensive reactive amino acid residues, which are then intricately linked with hemin using orthogonal click reactions, followed by protein refolding. This process not only retains the proteins’ structural integrity but expands proteins’ functionality. The most notable outcome of this methodology is the hemin@BAL variant, which demonstrated a remarkable 83.7% conversion rate in cyclopropanation reactions, far surpassing the capabilities of traditional hemin-based catalysis in water. This success highlights the significant role of protein structure in the ArMs’ activity and marks a substantial leap forward in chemical modification of proteins. Our findings suggest vast potentials of protein refolding approaches for ArMs across various catalytic applications, paving the way for future advancements in synthetic biology and synthetic chemistry.