Abstract: Enzymes harness a diverse array of nearly 40 protein-derived cofactors, often formed by irreversible amino acid crosslinking, to achieve remarkable catalytic feats.1 While the discovery of these cofactors is accelerating, understanding their intricate mechanisms remains a challenge due to the limitations of traditional mutagenesis. To address this, we have pioneered the use of site-specific incorporation of non-canonical amino acids (ncAAs), a powerful approach enabling precise dissection of cofactor biogenesis and function. This presentation will showcase our recent advances in elucidating the synthesis and function of key amino acid crosslinked cofactors in metalloenzymes. Specifically, we will discuss the formation of Cys-Tyr crosslinks in non-heme Fe-dependent2-3 and Cu-dependent enzymes4 and the unique Met+-Tyr-Trp cofactor in heme-dependent catalase-peroxidase (KatG).5 These studies provide the first mechanistic insight into Cys-Tyr cofactor biosynthesis in important enzyme families and have revealed a novel metal-mediated C–F bond cleavage,4,6-7 inspiring new avenues for catalyst design. Furthermore, we demonstrate the transformative potential of ncAA incorporation to precisely investigate the synthesis and function of these cofactors by probing specific C–C, C–S, and C–O bond formation, and the hydrogen atom transfer process in enzymatic catalysis.
Acknowledgment
This work is supported by NIH GM152982, and NSF CHE-2204225, and Welch Foundation AX-2110-20220331.
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