Purification and characterization of phenylacetate-coenzyme A ligase from a denitrifying Pseudomonas sp., an enzyme involved in the anaerobic degradation of phenylacetate.
The enzyme catalysing the first step in the anaerobic degradation pathway of phenylacetate was purified from a denitrifying Pseudomonas strain KB 740. It catalyses the reaction phenylacetate + CoA + ATP-->phenylacetyl-CoA + AMP + PPi and requires Mg2+. Phenylacetate-CoA ligase (AMP forming) was found in cells grown anaerobically with phenylacetate and nitrate. Maximal specific enzyme activity was 0.048 mumol min-1 x mg-1 protein in the mid-exponential growth phase. After 640-fold purification with 18% yield, a specific activity of 24.4 mumol min-1 mg-1 protein was achieved. The enzyme is a single polypeptide with Mr of 52 +/- 2 kDa. The purified enzyme shows high specificity towards the aromatic inducer substrate phenylacetate and uses ATP preferentially; Mn2+ can substitute for Mg2+. The apparent Km values for phenylacetate, CoA, and ATP are 60, 150, and 290 microM, respectively. The soluble enzyme has an optimum pH of 8.5, is insensitive to oxygen, but is rather labile and requires the presence of glycerol and/or phenylacetate for stabilization. The N-terminal amino acid sequence showed no homology to other reported CoA-ligases. The expression of the enzyme was studied by immunodetection. It is present in cells grown anaerobically with phenylacetate, but not with mandelate, phenylglyoxylate, benzoate; small amounts were detected in cells grown aerobically with phenylacetate.
Mohamed M el-S
Universitt Ulm, Germany.
Fuchs
G
G
D000249:Adenosine Monophosphate / Q000096:biosynthesis; D000595:Amino Acid Sequence; D000693:Anaerobiosis / Q000502:physiology; D001565:Benzoates / Q000378:metabolism; D019817:Benzoic Acid; D001673:Biodegradation, Environmental; D002853:Chromatography, Liquid; D003066:Coenzyme A Ligases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010648:Phenylacetates / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity
D001565:Benzoates; D010648:Phenylacetates; C026246:4-hydroxyphenylacetate; D000249:Adenosine Monophosphate; D019817:Benzoic Acid; D003066:Coenzyme A Ligases; C023914:phenylacetate - CoA ligase; C025136:phenylacetic acid