Purification and biochemical characterization of phenylacetyl-CoA ligase from Pseudomonas putida. A specific enzyme for the catabolism of phenylacetic acid.
A new enzyme, phenylacetyl-CoA ligase (AMP-forming) (PA-CoA ligase, EC 6.2.1-) involved in the catabolism of phenylacetic acid (PAA) in Pseudomonas putida is described and characterized. PA-CoA ligase was specifically induced by PAA when P. putida was grown in a chemically defined medium in which phenylacetic acid was the sole carbon source. Hydroxyl, methyl-phenylacetyl derivatives, and other PAA close structural molecules did not induce the synthesis of this enzyme and neither did acetic, butyric, succinic, nor fatty acids (greater than C5 atoms carbon length). PA-CoA ligase requires ATP, CoA, PAA, and MgCl2 for its activity. The maximal rate of catalysis was achieved in 50 mM HCl/Tris buffer, pH 8.2, at 30 degrees C and under these conditions, the Km calculated for ATP, CoA, and PAA were 9.7, 1.0, and 16.5 mM, respectively. The enzyme is inhibited by some divalent cations (Cu2+, Zn2+, and Hg2+) and by the sulfhydryl reagents N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and p-chloromercuribenzoate. PA-CoA ligase was purified to homogeneity (513-fold). It runs as a single polypeptide in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a molecular mass of 48 +/- 1 kDa. PA-CoA ligase does not use as substrate either 3-hydroxyphenylacetic, 4-hydroxyphenylacetic, or 3,4-dihydroxyphenylacetic acids and shows a substrate specificity different from other acyl-CoA-activating enzymes. The enzyme is detected in P. putida from the early logarithmic phase of growth and is repressed by glucose, suggesting that PA-CoA ligase is a specific enzyme involved in the utilization of PAA as energy source.
Martínez-Blanco
H
H
Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de León, Spain.
Reglero
A
A
Rodriguez-Aparicio
L B
LB
Luengo
J M
JM
D000596:Amino Acids / Q000032:analysis; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003066:Coenzyme A Ligases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D010648:Phenylacetates / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity; D013816:Thermodynamics; D014461:Ultracentrifugation
D000596:Amino Acids; D010648:Phenylacetates; D003066:Coenzyme A Ligases; C023914:phenylacetate - CoA ligase; C025136:phenylacetic acid