A Complex of 2-Hydroxyisocaproyl-Coenzyme A Dehydratase and its Activator from Clostridium difficile Stabilized by Aluminium Tetrafluoride-Adenosine Diphosphate

Title
A Complex of 2-Hydroxyisocaproyl-Coenzyme A Dehydratase and its Activator from Clostridium difficile Stabilized by Aluminium Tetrafluoride-Adenosine Diphosphate
Author(s)
김지회안토니오 피에릭[안토니오 피에릭]볼프강 부클[볼프강 부클]
Keywords
AMINO-ACID FERMENTATION; ACIDAMINOCOCCUS-FERMENTANS; 2-HYDROXYGLUTARYL-COA DEHYDRATASE; MECHANISTIC PROPOSAL; ANAEROBIC-BACTERIA; FE-PROTEIN; COA; LEUCINE; ACETATE
Issue Date
201004
Publisher
WILEY-V C H VERLAG GMBH
Citation
CHEMPHYSCHEM, v.11, no.6, pp.1307 - 1312
Abstract
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 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 rnol homodimeric activator, 1.0 mol heterodimeric dehydratise 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-lay 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.
URI
http://hdl.handle.net/YU.REPOSITORY/22595http://dx.doi.org/10.1002/cphc.200900876
ISSN
1439-4235
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생명공학부 > 생명공학부 > Articles
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