Human histone demethylase KDM6B can catalyse sequential oxidations

Abstract

Jumonji domain-containing demethylases (JmjC-KDMs) catalyse demethylation of Nε-methylated lysines on histones and play important roles in gene regulation. We report selectivity studies on KDM6B (JMJD3), a disease-relevant JmjC-KDM, using synthetic lysine analogues. The results unexpectedly reveal that KDM6B accepts multiple Nε-alkylated lysine analogues, forming alcohol, aldehyde and carboxylic acid products.

Fig. 1. Outline of N-demethylation catalysed by KDM6B. (a) Reaction scheme of KDM6B catalysis. Oxidation of an N-methyl C–H bond by KDM6B results in formation of an unstable hemiaminal, which fragments to form formaldehyde. (b) View from a crystal structure of KDM6B in complex with histone substrate (PDB ID: ; 4EZH). The methylated lysine-27 residue (red) protrudes towards the catalytic iron(ii) bound in the active site (for crystallographic purposes, nickel was substituted for iron). N-Oxalylglycine (green) is a structural analogue of 2-oxoglutarate. (c) Structures of lysine analogues used in this study. Note: the Lys(Me₂), Lys(Me/Et), Lys(Et₂), Lys(iPr) and Lys (Me/iPr) residues are likely protonated under the assay conditions used.

Fig. 2. Analysis of KDM6B catalysed oxidations using mass spectrometry. Mass spectra (MALDI) of incubations of KDM6B with (a) KQLATKAAR-Lys(Et₂)-SAPAT, (b) KQLATKAAR-Lys(Me/Et)-SAPAT, (c) KQLATKAAR-Lys(iPr)-SAPAT, (d) KQLATKAAR-Lys(Me/iPr)-SAPAT, and (e) KQLATKAAR-Orn(Me/iPr)-SAPAT. Mass spectra showing the unmodified peptides are overlaid (red). (f) MS time-course for the incubation of KDM6B with KQLATKAAR-Lys(Me/Et)-SAPAT over 30 minutes. No evidence of KDM6B-catalysed oxidation of the other substrate analogues given in Fig. 1 was observed.

Scheme 1. Summarised reactions of KDM6B with histone fragment peptides containing (a) Lys(Et₂), (b) Lys(Me/Et), (c) Lys(iPr), (d) Lys(Me/iPr), and (e) Orn(Me/iPr), at position 27.