Purification and properties of benzoate-coenzyme A ligase, a Rhodopseudomonas palustris enzyme involved in the anaerobic degradation of benzoate.
A soluble benzoate-coenzyme A (CoA) ligase was purified from the phototrophic bacterium Rhodopseudomonas palustris. Synthesis of the enzyme was induced when cells were grown anaerobically in light with benzoate as the sole carbon source. Purification by chromatography successively on hydroxylapatite, phenyl-Sepharose, and hydroxylapatite yielded an electrophoretically homogeneous enzyme preparation with a specific activity of 25 mumol/min per mg of protein and a molecular weight of 60,000. The purified enzyme was insensitive to oxygen and catalyzed the Mg2+ ATP-dependent formation of acyl-CoA from carboxylate and free reduced CoA, with high specificity for benzoate and 2-fluorobenzoate. Apparent Km values of 0.6 to 2 microM for benzoate, 2 to 3 microM for ATP, and 90 to 120 microM for reduced CoA were determined. The reaction product, benzoyl-CoA, was an effective inhibitor of the ligase reaction. The kinetic properties of the enzyme match the kinetics of substrate uptake by whole cells and confirm a role for benzoate-CoA ligase in maintaining entry of benzoate into cells as well as in catalyzing the first step in the anaerobic degradation of benzoate by R. palustris.
Geissler
J F
JF
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
Harwood
C S
CS
Gibson
J
J
D000693:Anaerobiosis; D001565:Benzoates / Q000378:metabolism*; D001673:Biodegradation, Environmental; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D012241:Rhodopseudomonas / Q000201:enzymology*; D013379:Substrate Specificity
D001565:Benzoates; D003066:Coenzyme A Ligases; C055455:benzoate coenzyme A ligase