Probing the Determinants of the Molecular Recognition in Metal-Dependent Deacetylase.

[摘要] Metal-dependent deacetylases catalyze a variety of essential reactions in nature However,most metallohydrolases can be activated by a number of divalent metal ions, such as Zn(II) or Fe(II). Human metal-dependent metalloenzyme, histone deacetylases 8 (HDAC 8), catalyze the deacetylation of acetylated lysine residues on histones and other protein substrates. Although it was known to be Zn(II)-metalloenzyme, there is growing evidence which indicates it is also activated by Fe(II). HDAC8 was more active with Fe(II) than Zn(II) in vitro, and exhibits Fe(II)-like activity in vivo, suggesting that many ;;Zn”-enzymes may utilize either Fe or Zn cofactors in vivo depending on cellular conditions. Here I measured the metal binding kinetics and thermodynamics for HDAC8 using newly developed fluorescence polarization assay.Next, the determinants governing the metal specificity of HDAC8 was explored. Alterations in second shell ligand environment significantly affect the reactivity (kcat/KM), binding affinity (koff and KD) for Zn/Fe-bound HDAC8 when compared to the wild-type. Monovalent binding, other metal binding sites in HDAC8, also had an effect on divalent metal binding by decreasing dissociation rate constant. Additionally, putative binding partner to HDAC8, poly (rC) binding protein, was investigated to determine the interaction between two proteins in vitro and in vivo. This finding revealed that two proteins bind to each other in a specific manner with μM affinity when apo-apo proteins interact. These all data suggest that HDAC8 is activated by either Zn or Fe based on various factors as a means of metal homeostasis.Finally, other deacetylase, UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC), was studied to generate novel potent inhibitor. LpxC catalyzes the committed step in Lipid A biosynthesis. Inhibitors of LpxC are targets for the development of antibacterials, since lipid A is essential for cell viability. For the development of more potent non-hydroxamate inhibitors, library of metal-binding fragments were screened against Escherichia Coli LpxC using high-throughpu mass spectrometry assay. A few potent hits (IC50 ≈ nM range) was identified. These results are important for understanding how best to inhibit the many clinically useful metalloprotein drug targets, as well as for understanding a potentially important new aspect of metal ion homeostasis and metalloprotein regulation.

[发布机构] University of Michigan

[效力级别] Biological Chemistry

[关键词] Metal selectivity of Histone Deacetylase 8;Biological Chemistry;Science;Chemical Biology