Probing the Charge and Conformational Requirements of JmjC Demethylases

Lysine residues are subjected to a wide range of post‐translational modifications including, but not limited to, Nɛ‐methylation(1), N ɛ ‐acetylation(2) and side chain hydroxylation(3). Dynamic methylation and demethylation of lysine residues of the histone 3 N ‐terminal tail plays an important role in epigenetic gene regulation(4). Demethylation of N ɛ ‐methyl lysine residues is catalyzed by demethylases, of which the largest family is the ferrous iron and 2‐oxoglutarate dependent demethylases (JmjC KDMs). The JmjC KDMs catalyze demethylation via initial hydroxylation of the N‐methyl groups and subsequent fragmentation of the resultant hemiaminal(5). A lack of efficient synthetic methods to modified lysine analogues has hampered biochemical, functional and inhibition studies on KDMs. We envisaged a synthetic strategy, employing olefin cross‐metathesis, to afford a range of lysine analogues in good yields from commercially available materials(6). A selection of unsaturated and saturated analogues were incorporated into substrate peptides and tested for activity with JmjC KDMs. Surprisingly, many analogues were substrates of JmjC KDMs, suggesting that KDM catalysis might be more promiscuous than presently perceived in vivo . Further, catalysis was only observed for analogues containing a positively charge ɛ‐amino group, implying that JmjC KDMs prefer positively charged substrates. Crystallographic analysis of KDM4A(7) and KDM7B(8) show that the substrate N ɛ ‐methylated lysine residue binds with different side chain conformations during catalysis. Despite this, a trans‐C‐4/C‐5 dehydrolysine substrate analogue, when incorporated into appropriate peptides, was a substrate for the KDM4 subfamily representatives and, surprisingly, KDM7B. Overall, the work has developed novel synthetic approaches to lysine derivatives, has identified novel KDM substrates and has provided insights into substrate (and by extension, inhibitor) binding. We envisage the work will inform future functional and inhibition studies on JmjC KDMs. Support or Funding Information This work was supported by funding from the Engineering and Physical Sciences Research Council, the Biotechnology and Biological Sciences Research Council, Cancer Research UK, UCB (GWL), the Systems Approaches to Biomedical Science Industrial Doctoral Centre (GWL) and the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (MM).