Using BpyAla to generate Copper Artificial Metalloenzymes: a catalytic and structural study

30 October 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

Artificial metalloenzymes (ArMs) have emerged as a promising avenue in the field of biocatalysis, offering new reactivity. However, their design remains challenging due to the limited understanding of their protein dynamics and how the introduced cofactors alter the protein scaffold structure. Here we present the structures and catalytic activity of novel copper ArMs capable of (R)- or (S)-stereoselective control, utilizing a steroid carrier protein (SCP) scaffold. To incorporate 2,2’-Bipyridine (Bpy) into SCP, two distinct strategies were employed: either Bpy was introduced as an unnatural amino acid (2,2’-bipyridin-5-yl)alanine (BpyAla) using amber stop codon expression or via bioconjugation of bromomethyl-Bpy to cysteine residues. The resulting ArMs proved to be effective at catalysing an enantioselective Friedel-Crafts reaction with SCP_Q111BpyAla achieving the best selectivity with an enantioselectivity of 72% ee (S). Interestingly, despite using the same protein scaffold, different attachment strategies for Bpy at the same residue (Q111) led to a switch in the enantiopreference of the ArM.

Keywords

Artificial Metalloenzymes
unnatural amino acids
Friedel-Crafts
non-canonical amino acids
protein structure

Supplementary materials

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Supporting Information A
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Additional experimental details, materials, methods and data.
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Supporting Information B
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Additional information on the computational studies.
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