LmrR: A Privileged Scaffold for Artificial Metalloenzymes

Area of Science:

• Biocatalysis and Enzyme Engineering
• Protein-Scaffolded Metalloenzymes
• Synthetic Biology

Background:

• Directed evolution enables enzyme creation but requires basal activity for new reactions.
• Artificial metalloenzymes (ARMs) offer a starting point for novel catalytic functions.
• The multidrug resistance regulator LmrR provides a versatile protein scaffold for ARM design.

Purpose of the Study:

• To detail the creation of artificial metalloenzymes (ARMs) for catalyzing new-to-nature reactions.
• To investigate the role of substrate binding and protein dynamics in ARM catalysis.
• To demonstrate the utility of the LmrR scaffold for diverse catalytic applications.

Main Methods:

• Rational design of ARMs based on the LmrR scaffold, utilizing its hydrophobic binding pocket.
• Introduction of catalytic metal complexes via covalent linkage, ligand modification, or supramolecular assembly.
• Application of expanded genetic code methodologies for in vivo biosynthesis of ARMs with unnatural amino acids.
• Computational studies to understand protein dynamics and guide redesign efforts.

Main Results:

• LmrR-based ARMs successfully catalyzed Friedel-Crafts alkylations and enantioselective cyclopropanations.
• Expanded genetic code methods and supramolecular assembly enabled efficient ARM creation and application.
• Computational redesign led to improved activity and selectivity in artificial hydratases.
• Structural dynamics of LmrR facilitate substrate access to buried catalytic centers.

Conclusions:

• LmrR is a privileged scaffold for developing diverse and highly active artificial metalloenzymes.
• LmrR-based ARMs can be created through various methods, including in vivo biosynthesis and supramolecular assembly.
• These ARMs are suitable for integration into biosynthetic pathways, paving the way for hybrid metabolism.