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Abstract Despite the unique reactivity of vitamin B₁₂and its derivatives, B₁₂‐dependent enzymes remain underutilized in biocatalysis. In this study, we repurposed the B₁₂‐dependent transcription factor CarH to enable non‐native radical cyclization reactions. An engineered variant of this enzyme, CarH*, catalyzes the formation γ‐ and δ‐lactams through either redox‐neutral or reductive ring closure with marked enhancement of reactivity and selectivity relative to the free B₁₂cofactor. CarH* also catalyzes an unusual spirocyclization by dearomatization of pendant arenes to produce bicyclic 1,3‐diene products instead of 1,4‐dienes provided by existing methods. These results and associated mechanistic studies highlight the importance of protein scaffolds for controlling the reactivity of B₁₂and expanding the synthetic utility of B₁₂‐dependent enzymes. 

The ability to site-selectively modify equivalent functional groups in a molecule has the potential to streamline syntheses and increase product yields by lowering step counts. Enzymes catalyze site-selective transformations throughout primary and secondary metabolism, but leveraging this capability for non-native substrates and reactions requires a detailed understanding of the potential and limitations of enzyme catalysis and how these bounds can be extended by protein engineering. In this review, we discuss representative examples of site-selective enzyme catalysis involving functional group manipulation and C–H bond functionalization. We include illustrative examples of native catalysis, but our focus is on cases involving non-native substrates and reactions often using engineered enzymes. We then discuss the use of these enzymes for chemoenzymatic transformations and target-oriented synthesis and conclude with a survey of tools and techniques that could expand the scope of non-native site-selective enzyme catalysis. 

Artificial metalloenzymes (ArMs) can combine the unique features of both metal complexes and enzymes by incorporating a cofactor within a protein scaffold. Herein, we describe a panel of ArMs constructed by covalently linking Ir( iii ) polypyridyl complexes into a prolyl oligopeptidase scaffold. Spectroscopic methods were used to examine how properties of the resulting ArMs are influenced by structural variation of the cyclometalated ligands and the protein scaffold. Visible light photocatalysis by these hybrid catalysts was also examined, leading to the finding that they catalyze inter/intra-molecular [2 + 2] photocycloaddition in aqueous solution. Low but reproducible enantioselectivity was observed using a cofactor that undergoes partial kinetic resolution upon bioconjugation within the ArM active site, showing the importance of scaffold/cofactor interactions for enabling selective ArM photocatalysis. Further evidence of the importance of cofactor/scaffold interactions was provided by analyzing native POP peptidase catalysis by the ArMs. Together, these studies show how Ir( iii )-based ArMs constitute a promising starting point for ongoing studies to control the stereoselectivity of EnT reactions by engineering substrate binding/activation motifs in POP. 

The corrinoid protein MtaC, which is natively involved in methyl transferase catalysis, catalyzes N -alkylation of aniline using ethyl diazoacetate. Our results show how the native preference of B 12 scaffolds for radical versus polar chemistry translates to non-native catalysis, which could guide selection of B 12 -dependent proteins for biocatalysis. MtaC also has high thermal stability and organic solvent tolerance, remaining folded even in pure methanol.