title: On the ATP-dependent activation of the radical enzyme (R)-2-hydroxyisocaproyl-CoA dehydratase.
abstract: Members of the 2-hydroxyacyl-CoA dehydratase enzyme family catalyze the β,α-dehydration of various CoA-esters in the fermentation of amino acids by clostridia. Abstraction of the nonacidic β-proton of the 2-hydroxyacyl-CoA compounds is achieved by the reductive generation of ketyl radicals on the substrate, which is initiated by the transfer of an electron at low redox potentials. The highly energetic electron needed on the dehydratase is donated by a [4Fe-4S] cluster containing ATPase, termed activator. We investigated the activator of the 2-hydroxyisocaproyl-CoA dehydratase from Clostridium difficile. The activator is a homodimeric protein structurally related to acetate and sugar kinases, Hsc70 and actin, and has a [4Fe-4S] cluster bound in the dimer interface. The crystal structures of the Mg-ADP, Mg-ADPNP, and nucleotide-free states of the reduced activator have been solved at 1.6-3.0 Å resolution, allowing us to define the position of Mg(2+) and water molecules in the vicinity of the nucleotides and the [4Fe-4S] cluster. The structures reveal redox- and nucleotide dependent changes agreeing with the modulation of the reduction potential of the [4Fe-4S] cluster by conformational changes. We also investigated the propensity of the activator to form a complex with its cognate dehydratase in the presence of Mg-ADP and Mg-ADPNP and together with the structural data present a refined mechanistic scheme for the ATP-dependent electron transfer between activator and dehydratase.
authors: Institut für Biologie, Strukturbiologie/Biochemie, Humboldt-Universität zu Berlin, D-10099 Berlin, Germany.
authors: Stefan H
authors: SH
authors: Knauer
authors: Wolfgang
authors: W
authors: Buckel
authors: Holger
authors: H
authors: Dobbek
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D003460:Crystallization; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000737:chemistry / Q000302:isolation & purification; D006836:Hydro-Lyases / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D020536:Enzyme Activators; D007506:Iron-Sulfur Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C559050:(R)-2-hydroxyisocaproyl-CoA dehydratase, Clostridium difficile; D006836:Hydro-Lyases