title: Inhibitor-mediated stabilization of the conformational structure of a histone deacetylase-like amidohydrolase.
abstract: Histone deacetylases are major regulators of eukaryotic gene expression. Not unexpectedly, histone deacetylases are among the most promising targets in cancer therapy. However, despite huge efforts in histone deacetylase inhibitor design, very little is known about the impact of histone deacetylase inhibitors on enzyme stability. In this study, the conformational stability of a well-established histone deacetylase homolog with high structural similarity (histone deacetylase-like amidohydrolase from Bordetella/Alcaligenes species FB188) was investigated using denaturation titrations and stopped-flow kinetics. Based on the results of these complementary approaches, we conclude that the interconversion of native histone deacetylase-like amidohydrolase into its denatured form involves several intermediates possessing different enzyme activities and conformational structures. The refolding kinetics has shown to be strongly dependent on Zn(2+) and to a lesser extent on K(+), which underlines their importance not only for catalytic function but also for maintaining the correct conformational structure of the enzyme. Two main unfolding processes of histone deacetylase-like amidohydrolase were differentiated. The unfolding occurring at submolar concentrations of the denaturant guanidine hydrochloride was not affected by inhibitor binding, whereas the unfolding at higher concentrations of guanidine hydrochloride was strongly affected. It was shown that the known inhibitors suberoylanilide hydroxamic acid and cyclopentylpropionyl hydroxamate are capable of stabilizing the conformational structure of histone deacetylase-like amidrohydrolase. Judging from the free energies of unfolding, the protein stability was increased by 9.4 and 5.4 kJ.mol(-1) upon binding of suberoylanilide hydroxamic acid and cyclopentylpropionyl hydroxamate, respectively.
authors.affiliation:  Department of Chemical Engineering and Biotechnology, Darmstadt University of Applied Sciences, Germany Institut für Molekulare Genetik und Praeparative Molekularbiologie, Institut fuer Mikrobiologie und Genetik, Goettingen, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Kern
authors.affiliation:  Department of Chemical Engineering and Biotechnology, Darmstadt University of Applied Sciences, Germany Institut für Molekulare Genetik und Praeparative Molekularbiologie, Institut fuer Mikrobiologie und Genetik, Goettingen, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Riester
authors.affiliation:  Department of Chemical Engineering and Biotechnology, Darmstadt University of Applied Sciences, Germany Institut für Molekulare Genetik und Praeparative Molekularbiologie, Institut fuer Mikrobiologie und Genetik, Goettingen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Hildmann
authors.affiliation:  Department of Chemical Engineering and Biotechnology, Darmstadt University of Applied Sciences, Germany Institut für Molekulare Genetik und Praeparative Molekularbiologie, Institut fuer Mikrobiologie und Genetik, Goettingen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schwienhorst
authors.affiliation:  Department of Chemical Engineering and Biotechnology, Darmstadt University of Applied Sciences, Germany Institut für Molekulare Genetik und Praeparative Molekularbiologie, Institut fuer Mikrobiologie und Genetik, Goettingen, Germany.
authors.forename: Franz-Josef
authors.initials: FJ
authors.lastname: Meyer-Almes
mesh_terms: D001884:Bordetella / Q000201:enzymology / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry*; D004795:Enzyme Stability; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D011188:Potassium / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases; D015032:Zinc; D011188:Potassium