Purification and some properties of component A of the 4-chlorophenylacetate 3,4-dioxygenase from Pseudomonas species strain CBS.
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.
D000596:Amino Acids / Q000032:analysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D049308:Dioxygenases; D007526:Isoelectric Point; D008970:Molecular Weight; D010105:Oxygenases / Q000302:isolation & purification*; D011549:Pseudomonas / Q000201:enzymology*
D000596:Amino Acids; D010105:Oxygenases; C043629:4-chlorophenylacetate 3,4-dioxygenase; D049308:Dioxygenases