Lysine Possesses the Optimal Chain Length for Histone Lysine Methyltransferase Catalysis

Abstract

Histone lysine methyltransferases (KMTs) represent an important class of epigenetic enzymes that play essential roles in regulation of gene expression in humans. Members of the KMT family catalyze the transfer of the methyl group from S-adenosylmethionine (SAM) to lysine residues in histone tails and core histones. Here we report combined MALDI-TOF MS experiments, NMR analyses and quantum mechanical/molecular dynamics studies on human KMT-catalyzed methylation of the most related shorter and longer lysine analogues, namely ornithine and homolysine, in model histone peptides. Our experimental work demonstrates that while lysine is an excellent natural substrate for KMTs, ornithine and homolysine are not. This study reveals that ornithine does not undergo KMT-catalyzed methylation reactions, whereas homolysine can be methylated by representative examples of human KMTs. The results demonstrate that the specificity of KMTs is highly sensitive to the side chain length of the residue to be methylated. The origin for the degree of the observed activities of KMTs on ornithine and homolysine is discussed.

Lysine Ethylation by Histone Lysine Methyltransferases

“…Due to the longer C−Se bond and higher reactivity of AdoSeEth, we hypothesized that KMTs might have the ability to catalyze the ethylation of ornithine, which is the lysine analogue shorter by one methylene group. In line with our earlier observation, we observed that G9a and GLP did not methylate H3Orn9, in the presence of AdoMet, within the limits of detection (Figure , top). Similarly, no G9a/GLP‐catalyzed ethylation of H3Orn9 was observed if AdoEth was used as a cosubstrate, even upon longer incubation times (Figures , middle, and S13).…”

“…We then performed comparative enzymatic assays for KMT‐catalyzed methylation (with AdoMet) and ethylation (with AdoEth and AdoSeEth) of synthetic histone peptides by using MALDI‐TOF MS, as recently described; histone H3 1–15 was used for studies with SETD7 (also known as KMT7), G9a (also known as KMT1C and EHMT2), and GLP (also known as KMT1D and EHMT1), and histone H4 13–27 was used for studies with SETD8 (also known as KMT5A). MALDI‐TOF MS data confirmed that human KMTs catalyzed nearly quantitative methylation of histone peptides in the presence of AdoMet: H3K4me, H4K20me, H3K9me3, and H3K9me3 were formed in the presence of SETD7, SETD8, G9a, and GLP, respectively (Figure , top).…”

“…This is in contrast with cases for the first and second methyl transfers in GLP in which the target lysine and methyllysine can be well aligned, respectively, with the transferable methyl group (Figure S24). The free energy profiles for the first and second ethylation reactions in Figure C demonstrate that, although GLP may catalyze the first ethylation reaction (with a free energy barrier of 18.3 kcal mol −1 , which is higher than that of 17.0 kcal mol −1 for the first methylation reaction), it is unlikely to be able to catalyze the second ethylation reaction with AdoEth because the free energy barrier becomes significantly higher (23.4 kcal mol −1 ). These results are in agreement with the observed monoethylated product, H3K9et, and lack of the diethylated product H3K9et2 in MALDI‐TOF assays (Figure D).…”

“…Expression and purification of KMTs : The four wild‐type human KMTs (SETD7, SETD8, G9a, and GLP) were expressed and purified as described previously . The wild‐type sequences of human methyltransferases were as follows: SETD8 (aa 186–352), SETD7 (aa 1–366), G9a (aa 913–1193), and GLP (aa 951–1235).…”

Methylation of geometrically constrained lysine analogues by histone lysine methyltransferases

“…6,8 Recent studies have expanded the knowledge concerning the biocatalytic activity of human KMTs via examining various lysine analogues as substrates, revealing that KMTs not only display a high degree of specificity towards lysine, but also to simple lysine analogues. [9][10][11][12][13][14][15][16] Despite the progress in understanding of the role of the substrate-binding pocket on KMT catalysis, it is presently unclear how does the flexible lysine side chain contributes to substrate efficiency. In the ternary complex, the histone lysine side chain typically exhibits the anti (zig-zag) conformation, presumably enabling an optimal positioning towards the SAM cosubstrate for efficient methyl transfer (Fig.…”