Purification and characterization of benzoyl-CoA ligase from a syntrophic, benzoate-degrading, anaerobic mixed culture.
The benzoyl-CoA ligase from an anaerobic syntrophic culture was purified to homogeneity. It had a molecular mass of around 420 kDa and consisted of seven or eight subunits of 58 kDa. The temperature optimum was 37-40 degrees C, the optimum pH around 8.0 and optimal activity required 50-100 mM TRIS-HCl buffer, pH 8.0 and 3-7 mM MgCl2; MgCl2 in excess of 10 mM was inhibitory. The activation energy for benzoate was 11.3 kcal/mol. Although growth occurred only with benzoate as a carbon source, the benzoyl-coenzyme A (CoA) ligase formed benzoyl-CoA esters with benzoate, 2-, 3- and 4-fluorobenzoate, picolinate, nicotinate and isonicotinate. Acetate was activated to acetyl-CoA by an acetyl-CoA synthetase. The Km values for benzoate, 2-, 3- and 4-fluorobenzoate were 0.04, 0.28, 1.48 and 0.32 mM, the Vmax values 1.05, 1.0, 0.7 and 0.98 units (U)/mg, respectively. For reduced CoA (CoA-SH) a Km of 0.07 mM and a Vmax of 1.05 U/mg and for ATP a Km of 0.16 mM and a Vmax of 1.08 U/mg was determined. Benzoate activation was inhibited by more than 6 mM ATP, presumably by pyrophosphate generation from ATP. The inhibition constant (Ki) for pyrophosphate was 5.7 mM. No homology of the N-terminal amino acid sequence with that of a 2-aminobenzoyl-CoA ligase of a denitrifying Pseudomonas sp. was found.
Auburger
G
G
Institut für Mikrobiologie, Universität Regensburg, Federal Republic of Germany.
Winter
J
J
D000106:Acetate-CoA Ligase / Q000378:metabolism; D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000378:metabolism*; D001565:Benzoates / Q000378:metabolism*; D019817:Benzoic Acid; D003066:Coenzyme A Ligases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011756:Diphosphates / Q000494:pharmacology; D008969:Molecular Sequence Data
D001565:Benzoates; D011756:Diphosphates; D019817:Benzoic Acid; D003066:Coenzyme A Ligases; D000106:Acetate-CoA Ligase; C055455:benzoate coenzyme A ligase