Histidine methyltransferase SETD3 methylates structurally diverse histidine mimics in actin

Abstract

Actin histidine Nτ‐methylation by histidine methyltransferase SETD3 plays an important role in human biology and diseases. Here, we report integrated synthetic, biocatalytic, biostructural, and computational analyses on human SETD3‐catalyzed methylation of actin peptides possessing histidine and its structurally and chemically diverse mimics. Our enzyme assays supported by biostructural analyses demonstrate that SETD3 has a broader substrate scope beyond histidine, including N‐nucleophiles on the aromatic and aliphatic side chains. Quantum mechanical/molecular mechanical molecular dynamics and free‐energy simulations provide insight into binding geometries and the free energy barrier for the enzymatic methyl transfer to histidine mimics, further supporting experimental data that histidine is the superior SETD3 substrate over its analogs. This work demonstrates that human SETD3 has a potential to catalyze efficient methylation of several histidine mimics, overall providing mechanistic, biocatalytic, and functional insight into actin histidine methylation by SETD3.

Substrate selectivity and inhibition of histidine JmjC hydroxylases MINA53 and NO66

“…Finally, we note the results with histidine substrate analogues in this work highlight the value of using of unnatural amino acid residues in examinations of biomolecular recognition and biocatalysis, as also used for probing lysine posttranslational modifications 28 and in studies on 2OG oxygenases acting on small molecules, e.g., in antibiotic biosynthesis. 29 Together with recent work on the histidine methyltransferase SETD3, 30 the current results demonstrate that histidine-modifying enzymes display clearly different substrate specificities.…”

“…, in antibiotic biosynthesis. 29 Together with recent work on the histidine methyltransferase SETD3, 30 the current results demonstrate that histidine-modifying enzymes display clearly different substrate specificities.…”

“…Rpl8-1-TriaA216, Rpl27a-1-TriaA39, Rpl8-4-TriaA216 and Rpl27a-4-TriaA39 were synthesised by the use of Fmoc-Dap(N3)-OH and Fmoc-N-(propargyl)-glycine-OH on His39 and His216 for Rpl8 and Rpl27a, respectively, as reported. 30 The click reagent (NaN 3 or TMS-acetylene, 45 equiv.) was dissolved in 300 mL Milli-Q water and then added to the Rpl27a 31-49 /Rpl8 205-224 peptide (1 equiv.)…”

Probing lysine posttranslational modifications by unnatural amino acids

“…141 SETD3 was found to catalyse methylation of structurally and chemically diverse histidine mimics in β-actin peptides. 142 Furthermore, a β-actin peptide carrying a methionine at position 73 was found to be a strong binder and inhibitor of SETD3. 143 To this end, methionine analogues were also incorporated at the position 73, leading to the discovery of several sub-micromolar inhibitors against the enzyme, containing hydrophobic unnatural amino acids.…”

Structural and Energetic Origin of Different Product Specificities and Activities for SETD3 and Its Mutants on the Methylation of the β-Actin H73K Peptide: Insights from a QM/MM Study

“…The experimental structures of SETD3 in complex with different ligands have been determined, and detailed structural analyses and biochemical experiments have been undertaken. ,− Deng et al have examined the mechanism of catalysis and the basis for the effects of certain mutations for the His73 methylation. Moreover, Hintzen et al have performed a combined synthetic, biochemical, structural, and computational investigation for the methylation reactions of structurally and chemically diverse mimics of the actin peptide catalyzed by SETD3. One experimental observation is that this enzyme also possesses a weak activity of methylation on the H73K β-actin peptide in which H73 is mutated into K73; for simplicity, the K73 peptide will be used in the future.…”