Using BpyAla to generate copper artificial metalloenzymes: a catalytic and structural study

Area of Science:

• Biocatalysis
• Organometallic Chemistry
• Protein Engineering

Background:

• Artificial metalloenzymes (ArMs) offer novel reactivity but their design is hindered by poor understanding of protein dynamics and cofactor-induced structural changes.
• Developing ArMs with predictable stereoselectivity is crucial for advancing biocatalysis.

Purpose of the Study:

• To design and characterize novel copper ArMs based on a steroid carrier protein (SCP) scaffold for enantioselective Friedel-Crafts reactions.
• To investigate how different cofactor (2,2'-bipyridine) incorporation strategies influence ArM structure, activity, and enantioselectivity.
• To elucidate the structural basis for switched enantioselectivity observed with different incorporation methods.

Main Methods:

• Synthesis of ArMs using two strategies: unnatural amino acid incorporation (BpyAla) and bioconjugation (bromomethyl-Bpy to cysteine).
• Assessing catalytic activity and enantioselectivity of the ArMs in a Friedel-Crafts reaction.
• Determining X-ray crystal structures of key ArMs with bound Cu(II) ions.
• Utilizing alanine scanning mutagenesis and computational analysis to understand active site differences.

Main Results:

• Novel copper ArMs were created using SCP scaffolds, achieving (R)- or (S)-stereoselective control in Friedel-Crafts reactions.
• The ArM SCP_Q111BpyAla demonstrated optimal performance with 72% enantioselectivity for the (S)-enantiomer.
• Different Bpy attachment strategies at the same residue (Q111) resulted in a switch in enantioselectivity.
• X-ray structures revealed distinct catalytic center orientations, explaining the enantioselectivity switch by differential substrate stabilization.

Conclusions:

• The study successfully designed copper ArMs with tunable enantioselectivity by varying cofactor incorporation methods.
• Structural insights reveal how subtle changes in the active site's catalytic center orientation dictate enantiomeric preference.
• Integrating structural, catalytic, and computational approaches is vital for rational ArM design and advancing biocatalysis.