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Publication Abstract from PubMed

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 major challenge due to the absence of spatial control over chemical processes. Herein, through X-ray crystallography and cryo-electron microscopy structural studies, we describe the rational design of a nature-inspired, noncanonical amino-acid-incorporated, human ferritin-based metalloenzyme that is capable of introducing site-specific post-translational modifications (PTMs) to histidine in peptides and proteins. 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, moreover, site-specific post-translational modifications to peptides and proteins, which may further enable reactions to be carried out in vivo.

Site-Specific Histidine Aza-Michael Addition in Proteins Enabled by a Ferritin-Based Metalloenzyme.,Tsou JC, Tsou CJ, Wang CH, Ko AA, Wang YH, Liang HH, Sun JC, Huang KF, Ko TP, Lin SY, Wang YS J Am Chem Soc. 2024 Nov 5. doi: 10.1021/jacs.4c14446. PMID:39499210^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.