Enzymatic and computational studies on the simplest lysine analogues that possess a modified main chain demonstrate that the lysine's backbone contributes significantly to functional KMT binding and catalysis.

Methylation of lysine residues in histone proteins is catalyzed by S-adenosylmethionine (SAM)dependent histone lysine methyltransferases (KMts), a genuinely important class of epigenetic enzymes of biomedical interest. Here we report synthetic, mass spectrometric, nMR spectroscopic and quantum mechanical/molecular mechanical (QM/MM) molecular dynamics studies on KMtcatalyzed methylation of histone peptides that contain lysine and its sterically demanding analogs. our synergistic experimental and computational work demonstrates that human KMts have a capacity to catalyze methylation of slightly bulkier lysine analogs, but lack the activity for analogs that possess larger aromatic side chains. overall, this study provides an important chemical insight into molecular requirements that contribute to efficient KMT catalysis and expands the substrate scope of KMTcatalyzed methylation reactions. Posttranslational modifications on histone proteins regulate the structure and function of human chromatin 1-3. Well-established examples include lysine acetylation, which is linked with the transcriptionally active region of human genome, and lysine methylation, which is associated with gene activation and suppression, depending on the histone sequence and methylation state 4,5. Histone lysine methylation is catalyzed by S-adenosylmethionine (SAM)-dependent histone lysine methyltransferases (KMTs), and can lead to a formation of monomethyllysine (Kme), dimethyllysine (Kme2) and trimethyllysine (Kme3) 6,7. It is generally believed that the methylation state depends on the constitution of the KMT active site (Fig. 1a) 8. With the exception of DOT1L, all members of KMT family possess the SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) domain 9-11. Structural analyses of KMTs complexed with histone peptide/methylated peptide and S-adenosylhomocysteine product (SAH) revealed that the lysine side chain occupies a narrow, hydrophobic channel, typically comprised of side chains of several tyrosine and phenylalanine residues (Fig. 1b) 8. The positioning of the lysine's N ε amino group towards the electrophilic methyl group of the SAM cosubstrate results in an efficient methyl transfer via S N 2 reaction 12,13. Recent examinations of lysine analogs as substrates for human histone lysine methyltransferases revealed that KMTs possess a high degree of specificity for lysine residues. Enzymatic assays employing MALDI-TOF MS verified that human KMTs preferentially catalyze methylation of lysine residues with L-stereochemistry over D-stereochemistry 14. Combined experimental and computational studies on histone peptides that bear lysine analogs of different chain length revealed that lysine exhibits an optimal chain length for KMT-catalyzed methylation 15 , and that the enzymatic methylation is limited to N-nucleophiles 16. Members of KMTs were also found to catalyze methylation of the cysteine-derived γ-thialysine on intact histones and histone peptides 17,18. Substrate capturing studies using the genetically encoding photo-lysine showed th...