Purification and characterization of benzoate-coenzyme A ligase and 2-aminobenzoate-coenzyme A ligases from a denitrifying Pseudomonas sp.
The enzymes catalyzing the formation of coenzyme A (CoA) thioesters of benzoate and 2-aminobenzoate were studied in a denitrifying Pseudomonas sp. anaerobically grown with these aromatic acids and nitrate as sole carbon and energy sources. Three different rather specific aromatic acyl-CoA ligases, E1, E2, and E3, were found which catalyze the formation of CoA thioesters of benzoate, fluorobenzoates, and 2-aminobenzoate. ATP is cleaved into AMP and pyrophosphate. The enzymes were purified, their N-terminal amino acid sequences were determined, and their catalytic and molecular properties were studied. Cells anaerobically grown on benzoate and nitrate contain one CoA ligase (AMP forming) for benzoic acid (E1). It is a homodimer of Mr 120,000 which prefers benzoate as a substrate but shows some activity also with 2-aminobenzoate and fluorobenzoates, although with lower Km. Cells anaerobically grown on 2-aminobenzoate and nitrate contain three different CoA ligases for aromatic acids. The first one is identical with benzoate-CoA ligase (E1). The second enzyme is a 2-aminobenzoate-CoA ligase (E2). It is a monomer of Mr 60,000 which prefers 2-aminobenzoate but also activates benzoate, fluorobenzoates and, less effectively, 2-methylbenzoate, with lower affinities to the latter substrates. The enzymes E1 and E2 have similar activity levels; a third minor CoA ligase activity is due to a different 2-aminobenzoate-CoA ligase. The enzyme (E3) is a monomer of Mr, 65,000 which 2-aminobenzoate pathway (U. Altenschmidt, C. Eckerskorn, and G. Fuchs, Eur. J. Biochem. 194:647-653, 1990); apparently, it is not completely repressed under anaerobic conditions and therefore also is induced to a small extent by 2-aminobenzoate under anaerobic growth conditions.
Altenschmidt
U
U
Angewandte Mikrobiologie, University of Ulm, Germany.
Oswald
B
B
Fuchs
G
G
D000595:Amino Acid Sequence; D002384:Catalysis; D002853:Chromatography, Liquid; D003066:Coenzyme A Ligases / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity
C070685:2-aminobenzoate coenzyme A ligase; D003066:Coenzyme A Ligases; C055455:benzoate coenzyme A ligase