On the function of the internal cavity of histone deacetylase protein 8: R37 is a crucial residue for catalysis

Biochemical studies reveal that a conserved arginine residue (R37) at the centre of the 14 Å internal cavity of histone deacetylase (HDAC) 8 is important for catalysis and acetate affinity. Computational studies indicate that R37 forms multiple hydrogen bonding interactions with the backbone carbonyl oxygen atoms of two conserved glycine residues, G303 and G305, resulting in a ‘closed’ form of the channel. One possible rationale for these data is that water or product (acetate) transit through the catalytically crucial internal channel of HDAC8 is regulated by a gating interaction between G139 and G303 tethered in position by the conserved R37.     

Comparison of methods for determination of vitamin B12 in microbial material

Three different methods for the measurement of vitamin B12 were compared: two spectrophotometric methods and an HPLC one. When the pure vitamin was used, the results obtained using all three methods were similar, but when samples from microbial material were used, the results were different. The HPLC method could distinguish the true vitamin B12 from the different vitamin B12 analogues whereas the spectrophotometric methods could not.     

Thermodynamics of ligand binding to histone deacetylase like amidohydrolase from Bordetella/Alcaligenes

Thermodynamic studies on ligand–protein binding have become increasingly important in the process of drug design. In combination with structural data and molecular dynamics simulations, thermodynamic studies provide relevant information about the mode of interaction between compounds and their target proteins and therefore build a sound basis for further drug optimization. Using the example of histone deacetylases (HDACs), particularly the histone deacetylase like amidohydrolase (HDAH) from Bordetella/Alcaligenes, a novel sensitive competitive fluorescence resonance energy transfer‐based binding assay was developed and the thermodynamics of interaction of both fluorescent ligands and inhibitors to histone deacetylase like amidohydrolase were investigated. The assay consumes only small amounts of valuable target proteins and is suitable for fast kinetic and mechanistic studies as well as high throughput screening applications. Binding affinity increased with increasing length of aliphatic spacers (n = 4–7) between the hydroxamate moiety and the dansyl head group of ligand probes. Van't Hoff plots revealed an optimum in enthalpy contribution to the free energy of binding for the dansyl‐ligand with hexyl spacer. The selectivity in the series of dansyl‐ligands against human class I HDAC1 but not class II HDACs 4 and 6 increased with the ratio of ΔH⁰/ΔG⁰. The data clearly emphasize the importance of thermodynamic signatures as useful general guidance for the optimization of ligands or rational drug design. Copyright © 2014 John Wiley & Sons, Ltd.