Purification and some properties of component A of the 4-chlorophenylacetate 3,4-dioxygenase from Pseudomonas species strain CBS

• Published in: The Journal of biological chemistry 1986-09, Vol.261 (27), p.12883-12888
• Main Authors: Markus, A, Krekel, D, Lingens, F
• Format: Article
• Language: English
• Subjects: Amino Acids - analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; Dioxygenases; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Isoelectric Point; Molecular Weight; Oxidoreductases; Oxygenases - isolation & purification; Pseudomonas - enzymology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)67175-3

Pseudomonas sp. strain CBS 3 possesses a two-component enzyme system which converts 4-chlorophenylacetate to 3,4-dihydroxyphenylacetate by the incorporation of 2 atoms of molecular oxygen. Component A of this enzyme system was purified to homogeneity by a 5-step procedure. After the last purification step the enzyme was homogeneous in analytical and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the native protein was determined to be 140,000 by Sephadex G-200 and 144,000 by analytical ultracentrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that component A consists of three identical subunits with a molecular weight determined to range between 46,000 and 52,000. The isoelectric point was estimated to be 5.0. Component A shows an intensive red-brown color, and in the oxidized state it exhibits a visible absorption spectrum with a maximum at 458 nm and a shoulder at 560 nm. By reduction with sodium dithionite a new peak with a maximum at 518-520 nm is observed. The enzyme contains iron (1.6-1.8 mol/subunit) and acid-labile sulfide (1.6-1.9 mol/subunit) which suggests that component A is an iron-sulfur protein.