title: Purification of Pseudomonas putida acyl coenzyme A ligase active with a range of aliphatic and aromatic substrates.
abstract: Acyl coenzyme A (acyl-CoA) ligase (acyl-CoA synthetase [ACoAS]) from Pseudomonas putida U was purified to homogeneity (252-fold) after this bacterium was grown in a chemically defined medium containing octanoic acid as the sole carbon source. The enzyme, which has a mass of 67 kDa, showed maximal activity at 40 degrees C in 10 mM K2PO4H-NaPO4H2 buffer (pH 7.0) containing 20% (wt/vol) glycerol. Under these conditions, ACoAS showed hyperbolic behavior against acetate, CoA, and ATP; the Kms calculated for these substrates were 4.0, 0.7, and 5.2 mM, respectively. Acyl-CoA ligase recognizes several aliphatic molecules (acetic, propionic, butyric, valeric, hexanoic, heptanoic, and octanoic acids) as substrates, as well as some aromatic compounds (phenylacetic and phenoxyacetic acids). The broad substrate specificity of ACoAS from P. putida was confirmed by coupling it with acyl-CoA:6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum to study the formation of several penicillins.
authors: Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de León, Spain.
authors: M
authors: M
authors: Fernández-Valverde
authors: A
authors: A
authors: Reglero
authors: H
authors: H
authors: Martinez-Blanco
authors: J M
authors: JM
authors: Luengo
mesh_terms: D002210:Caprylates; D003066:Coenzyme A Ligases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003470:Culture Media / Q000737:chemistry; D004790:Enzyme Induction; D010648:Phenylacetates; D016958:Pseudomonas putida / Q000201:enzymology*; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D000098859:Long-Chain-Fatty-Acid-CoA Ligase
chemical_list: D002210:Caprylates; D003470:Culture Media; D010648:Phenylacetates; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D003066:Coenzyme A Ligases; C090799:FAA2 protein, S cerevisiae; D000098859:Long-Chain-Fatty-Acid-CoA Ligase; C025136:phenylacetic acid