Histidine Ethylation by Histidine Methyltransferases SETD3 and METTL9.

AdoMet-dependent histidine methyltransferases catalyze regioselective methylation of histidine residues in proteins. N^τ-Methylation of His73 in β-actin is catalyzed by histidine methyltransferase SETD3, and represents a unique post-translational modification involved in the regulation of actin polymerization. Likewise, N^π-methylation of His375 in zinc transporter SLC39A5 is catalyzed by histidine methyltransferase METTL9, thereby modulating zinc-binding properties of SLC39A5. Here, we report biomolecular studies on the ability of human SETD3 and METTL9 to catalyze the histidine ethylation reaction beyond methylation. Combined synthetic, biocatalytic and computational analyses employing synthetic or in situ formed AdoMet analogs AdoEth and AdoSeEth reveal that AdoMet is the most efficient cosubstrate; however, SETD3 and METTL9 also have the capacity to catalyze ethylation of histidine in β-actin and SLC39A5 peptides, respectively. Computational analyses support the experimental observations and provide the structural origin for more efficient histidine methylation than ethylation reaction. This work provides an insight into the molecular requirements for histidine methyltransferase-catalyzed histidine methylation and most related ethylation reactions on the N^τ- and N^π-positions in the imidazole ring, the knowledge important for functional assignment and design of chemical probes targeting histidine methyltransferases.

© 2026 The Author(s). ChemBioChem published by Wiley‐VCH GmbH.