A complex of 2-hydroxyisocaproyl-coenzyme A dehydratase and its activator from Clostridium difficile stabilized by aluminium tetrafluoride-adenosine diphosphate.
The dehydration of 2-hydroxyisocaproyl-CoA to isocaprenoyl-CoA is the chemically most demanding step in the reduction of leucine to isocaproate by Clostridium difficile, because the beta-hydrogen of the substrate is not acidic (pK(a) ca. 40). A two-component system, composed of a homodimeric activator and an heterodimeric dehydratase, catalyses this unusual alpha,beta-elimination of water. The reduced activator transfers an electron from its [4Fe-4S](+) cluster to that of the dehydratase in an ATP-dependent manner, similar to the iron protein of nitrogenase. Here we show that AlF(4)(-) x ADP traps the interaction of the activator with the dehydratase by forming a stable complex containing 1.0 mol homodimeric activator, 1.0 mol heterodimeric dehydratase and 1.2 mol ADP. The complex (148 kDa) was isolated by size exclusion chromatography, affinity chromatography using the Strep-tag at the activator, or most conveniently by ultrafiltration (100 kDa cut off membrane). Kinetic and EPR-spectroscopic experiments revealed that the complex formation proceeds much slower than the activation but in an almost irreversible manner. The isolated complex is devoid of any activity, because the dehydratase is in the oxidized form whereas the activator remains in the reduced state due to the presence of dithionite.
Kim
Jihoe
J
Fachbereich Biologie, Philipps Universität, 35032 Marburg, Germany.
Pierik
Antonio J
AJ
Buckel
Wolfgang
W
D000244:Adenosine Diphosphate / Q000737:chemistry*; D017607:Aluminum Compounds / Q000737:chemistry*; D016360:Clostridioides difficile / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D005459:Fluorides / Q000737:chemistry*; D006836:Hydro-Lyases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet
D017607:Aluminum Compounds; C050992:tetrafluoroaluminate; D000244:Adenosine Diphosphate; D006836:Hydro-Lyases; D005459:Fluorides