Streptavidin (Sav)-Based Artificial Metalloenzymes: Cofactor Design Considerations and Large-Scale Expression of Host Protein Variants

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

“…15,16 More synthetic approaches to site-specific incorporation of metal complexes include the utilisation of supramolecular binding, with several different tethered transition metal complex systems explored to date, the most notable being the biotin-streptavidin (Sav) system. 17 Bioconjugation of metal complexes via covalent attachment with unique reactive amino acid residues such as cysteine or azidophenylalanine is another widely used approach to prepare ArMs. 18 In vivo ligand incorporation offers an attractive alternative where metal-binding unnatural amino acids such as (2,2′-bipyridin-5-yl)alanine (BpyAla) can be selectively introduced to the protein scaffold directly using genetic code expansion technologies, allowing for more streamlined enzyme engineering.…”

“…The most prominent examples are the LmrR scaffold, which allows supramolecular, bioconjugation, and incorporation of unnatural amino acids, 21 and Sav, which has been explored in dative coordination, 22 alongside the more common supramolecular approach. 17 Despite these advances, few comparative studies have been conducted which included direct comparisons of the modification strategy for site-selective metal coordination. This makes it difficult to discern the role of protein modification upon ArM reactivity and predict the most effective route to ArM assembly for a desired application.…”