Oral Presentation 21st International Conference on Biological Inorganic Chemistry 2025

Site-specific histidine aza-Michael addition in proteins enabled by a ferritin-based metalloenzyme (119680)

Jo-Chu Tsou 1 , Chun-Ju Tsou 1 2 , An-Li A. Ko 1 , Yi-Hui Wang 1 , Huan-Hsuan Liang 1 2 , Jia-Cheng Sun 1 , Kai-Fa Huang 1 , Tzu-Ping Ko 1 , Yane-Shih Wang 1 2
  1. Institute of Biological Chemistry, Academia Sinica, Taipei, TAIPEI, Taiwan
  2. Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan

Histidine modifications of proteins are broadly based on chemical methods triggering N-substitution reactions such as aza-Michael addition at histidine’s moderately nucleophilic imidazole side chain. While recent studies have demonstrated chemoselective, histidine-specific modifications by further exploiting imidazole’s electrophilic reactivity to overcome interference from the more nucleophilic lysine and cysteine, achieving site-specific histidine modifications remains a significant challenge due to the absence of spatial control over chemical processes1. Herein, through X-ray crystallography and cryo-electron microscopy structural studies, we describe the rational design of a nature-inspired, noncanonical amino-acid-incorporated2, human ferritin-based3 copper metalloenzyme that is capable of introducing site-specific post-translational modifications (PTMs) to histidine in peptides and proteins4. Specifically, chemoenzymatic aza-Michael additions on single histidine residues were carried out on eight protein substrates ranging from 10 to 607 amino acids, including the insulin peptide hormone. By introducing an insulin-targeting peptide into our metalloenzyme, we further directed modifications to be carried out site-specifically on insulin’s B-chain histidine 5. The success of this biocatalysis platform outlines a novel approach in introducing residue- and site-specific post-translational modifications to peptides and proteins, which may further enable reactions to be carried out in vivo.

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