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A Complex of 2‐Hydroxyisocaproyl‐Coenzyme A Dehydratase and its Activator from Clostridium difficile Stabilized by Aluminium Tetrafluoride‐Adenosine Diphosphate
Author(s) -
Kim Jihoe,
Pierik Antonio J.,
Buckel Wolfgang
Publication year - 2010
Publication title -
chemphyschem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.016
H-Index - 140
eISSN - 1439-7641
pISSN - 1439-4235
DOI - 10.1002/cphc.200900876
Subject(s) - dehydratase , chemistry , azotobacter vinelandii , activator (genetics) , biochemistry , dithionite , ternary complex , enzyme , stereochemistry , nitrogenase , organic chemistry , nitrogen fixation , nitrogen , gene
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 β‐hydrogen of the substrate is not acidic (p K a ca. 40). A two‐component system, composed of a homodimeric activator and an heterodimeric dehydratase, catalyses this unusual α,β‐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 − × 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.