title: Caffeate respiration in the acetogenic bacterium Acetobacterium woodii: a coenzyme A loop saves energy for caffeate activation.
abstract: The anaerobic acetogenic bacterium Acetobacterium woodii couples reduction of caffeate with electrons derived from molecular hydrogen to the synthesis of ATP by a chemiosmotic mechanism with sodium ions as coupling ions. Caffeate is activated to caffeyl coenzyme A (caffeyl-CoA) prior to its reduction, and the caffeate reduction operon encodes an ATP-dependent caffeyl-CoA synthetase that is thought to catalyze the initial caffeate activation. The operon also encodes a potential CoA transferase, the product of carA, which was thought to be involved in subsequent ATP-independent caffeate activation. To prove the proposed function of carA, we overproduced its protein in Escherichia coli and then purified it. Purified CarA drives the formation of caffeyl-CoA from caffeate with hydrocaffeyl-CoA as the CoA donor. The dependence of the reaction on caffeate and hydrocaffeyl-CoA followed Michaelis-Menten kinetics, with apparent K(m) values of 75 ± 5 μM for caffeate and 8 ± 2 μM for hydrocaffeyl-CoA. The enzyme activity had broad ranges of pH and temperature optima. In addition to being able to use caffeate, CarA could use p-coumarate and ferulate but not cinnamate, sinapate, or p-hydroxybenzoate as a CoA acceptor. Neither acetyl-CoA nor butyryl-CoA served as the CoA donor for CarA. The enzyme uses a ping-pong mechanism for CoA transfer and is the first classified member of a new subclass of family I CoA transferases that has two catalytic domains on one polypeptide chain. Apparently, CarA catalyzes an energy-saving CoA loop for caffeate activation in the steady state of caffeate respiration.
authors: Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt am Main, Frankfurt, Germany.
authors: Verena
authors: V
authors: Hess
authors: José M
authors: JM
authors: González
authors: Anutthaman
authors: A
authors: Parthasarathy
authors: Wolfgang
authors: W
authors: Buckel
authors: Volker
authors: V
authors: Müller
mesh_terms: D045855:Acetobacterium / Q000201:enzymology* / Q000378:metabolism*; D002109:Caffeic Acids / Q000378:metabolism*; D003065:Coenzyme A / Q000378:metabolism*; D004734:Energy Metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D053858:Metabolic Networks and Pathways; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002109:Caffeic Acids; D011994:Recombinant Proteins; D003065:Coenzyme A; C040048:caffeic acid