Dynamic methylations and demethylations of histone lysine residues are important 
for gene regulation and are facilitated by histone methyltransferases and 
histone demethylases (HDMs). KDM5B/Jarid1B/PLU1 is an H3K4me3/me2-specific 
lysine demethylase belonging to the JmjC domain-containing family of histone 
demethylases (JHDMs). Several studies have linked KDM5B to breast, prostate and 
skin cancer, highlighting its potential as a drug target. However, most 
inhibitor studies have focused on other JHDMs, and inhibitors for KDM5B remain 
to be explored. Here, we report the expression, purification and 
characterization of the catalytic core of recombinant KDM5B (ccKDM5B, residues 
1-769). We show that ccKDM5B, recombinantly expressed in insect cells, 
demethylates H3K4me3 and H3K4me2 in vitro. The kinetic characterization showed 
that ccKDM5B has an apparent Michaelis constant (K(m) (app) ) value of 0.5 μm 
for its trimethylated substrate H3(1-15)K4me3, a considerably increased apparent 
substrate affinity than reported for related HDMs. Despite the presence of a PHD 
domain, the catalytic activity was not affected by additional methylation at the 
H3K9 position, suggesting that in vitro chromatin cross-talk between H3K4 and 
H3K9 does not occur for ccKDM5B. Inhibition studies of ccKDM5B showed both in 
vitro and in cell inhibition of ccKDM5B by 2,4-pyridinedicarboxylic acid 
(2,4-PDCA) with a potency similar to that reported for the HDM KDM4C. 
Structure-guided sequence alignment indicated that the binding mode of 2,4-PDCA 
is conserved between KDM4A/C and KDM5B.