Fig. 3: Hst2 is a histone debenzoylase.

a Fluorescence quantitative western blot analysis comparing the Kbz and Kac levels in WT (BY4742) and ten HDAC-deletion strains. The fluorescence signals from mutant strains were normalized by setting the WT fluorescence signal to 1.0 and were labeled in the image. The H3* represents clipping H3. Source data are provided in the Source Data file. b Debenzoylase activity of Hst2_FL protein (2 μM) with H3K9bz peptide (10 μM) shown by the representative MALDI-TOF spectra at 0, 5, and 30 min. The peaks for H3K9bz (bz, m/z 2,337) and unmodified (un, m/z 2,233) products are labeled. c Deacetylase activity of Hst2_FL protein (0.5 μM) with H3K9ac peptide (10 μM) shown by the representative MALDI-TOF spectra at 0, 5, and 30 min. The peaks for H3K9ac (ac, m/z 2,737) and unmodified (un, m/z 2695) products are labeled. d The Michaelis-Menten plot for Hst2 fixed at 0.1 μM with varied H3K9bz peptide concentrations. Data are presented as mean ± SD, n = 3 biological independent measurements. Source data for panels (d, e) are provided in the Source Data file. e The Michaelis–Menten plots for Hst2 fixed at 0.05 μM with varied H3K9ac peptide concentrations. Data are presented as mean ± SD, n = 3 biological independent measurements. f Hst2-mediated debenzoylation reaction monitored by time-resolved MALDI-TOF mass spectrometry. The Y-axis represents the percentage of Kbz peptide in total peptides calculated from the MALDI-TOF spectra. Data are presented as mean ± SD, n = 3 biological independent measurements. Source data are provided in the Source Data file. g ITC binding curves for Hst2 interaction with different Kbz peptides. h Sequence alignment of benzoylated H3 peptides used in the current study. The alignment is centered on the target lysine (yellow). Positively charged R/K residues are shown in blue. The dissociation constants (K_d) and enthalpy changes (ΔH) derived from the ITC assay (panel g) are shown.