Mechanistic Studies on Histone Demethylases and Related Enzymes

Histone lysyl demethylases (2OG demethylases) are a family of nuclear proteins that catalyse the demethyation of Nε-methylated lysines on histone tails. 2OG demethylases belong to the 2-oxoglutarate and Fe(II) dependent dioxygenase superfamily, which utilise molecular oxygen to oxidise a wide variety of cellular substrates. The methylation states of histone lysines play crucial roles in regulating gene transcription, and consequently, the 2OG demethylases have been identified as important regulators of chromatin. Therefore, the understanding of the mechanisms and cellular activities of the demethylases is of considerable interest in the context of chromatin biology, and also for medicinal chemistry. Work in this thesis has focused upon investigating the mechanisms and reactivities of 2OG demethylases in vitro. NMR spectroscopy techniques were used to monitor demethylation catalysed by the 2OG demethylase JMJD2E, which resulted in the acquisition of kinetic parameters. Also, the use of a 13C-labelled substrate during NMR analysis enabled the first direct detection of enzymatically-produced formaldehyde. Studies with methylated lysine analogues using mass spectrometry and NMR methods revealed that many 2OG demethylases are capable of oxidising multiple substrates. One such substrate, Nε-methylisopropyllysine, was found to be hydroxylated by JMJD2E, providing strong evidence that 2OG demethylase-catalysed lysyl demethylation proceeds via hydroxylation. Studies with the lysine analogues prompted investigations with methylated arginine peptides; unexpectedly, three 2OG demethylases were observed to demethylate methylated arginines in both histone peptide variants and at known methylarginine sites in histone peptides. These findings indicate that 2OG demethyases may have diversified functions in cells, and are capable of accepting substrates besides methylated lysines. Investigations with point-substituted variants of the 2OG demethylase JMJD2A revealed new insights into the role of lysine-241 during catalysis. Specifically, the proposed role of lysine-241 in oxygen binding was discredited, with evidence suggesting that lysine-241 is likely to be involved in binding the substrate in the active site. Mass spectrometry experiments with the 2OG demethylase FBXL11 identified hydrolysis of histone peptides during incubation with the protein. Further analyses using mass spectrometry revealed that a metalloprotease was the likely catalyst for histone cleavage, indicating that optimised expression and purification of FBXL11 is required for quantitative assays. Studies with deuterated methyllysine substrates revealed a small kinetic isotope effect during 2OG demethylase-catalysed demethylation, indicating that the hydroxylation step during catalysis is partially rate determining. Finally, work was concentrated on investigating the potential metabolism of formaldehyde released during histone demethylation. NMR studies were conducted monitoring the non-enzymatic reaction of glutathione and formaldehyde; these revealed two novel adduct species that may be formed in cellular environments. The function and mechanism of a putative formaldehyde activating enzyme (GFA) was then investigated. These studies showed a limited effect of GFA upon formaldehyde reactivity, suggesting that GFA is not involved in formaldehyde metabolism. In summary, this work has revealed many new insights into both the substrate specificities and catalytic mechanisms of 2OG demethylases and has provided extensive studies probing potential formaldehyde metabolism. These investigations should form the basis of many new research areas on 2OG demethylases and related enzymes.