Purification and characterization of phthalate oxygenase and phthalate oxygenase reductase from Pseudomonas cepacia.
An enzymatic system has been isolated that catalyzes dihydroxylation of phthalate to form 1,2-dihydroxy-4,5-dicarboxy-3,5-cyclohexadiene with consumption of NADH and O2. This system is comprised of two proteins: a flavo-iron-sulfur protein with NADH-dependent oxidoreductase activity and a nonheme iron protein with oxygenase activity. Phthalate oxygenase is a large (approximately 217 kDa) protein composed of apparently identical 48-kDa monomers. The active enzyme has one Rieske-type [2Fe-2S] center and one mononuclear iron/monomer. Removal of the mononuclear iron by incubation with EDTA or with o-phenanthroline inhibits oxygenation; ferrous ion completely restores activity. No other metals are effective. Phthalate oxygenase is specific for phthalate or other closely related compounds. However, only phthalate is tightly coupled to NADH oxidation and O2 consumption with a stoichiometry of 1:1:1. Phthalate oxygenase is chemically competent to oxygenate phthalate when artificially supplied with reducing equivalents and O2. Phthalate oxygenase reductase is required, however, for efficient catalytic activity. The reductase is a monomeric 34-kDa flavo-iron-sulfur protein containing FMN and a plant-ferredoxin-type [2Fe-2S] center in a 1:1 ratio. Phthalate oxygenase reductase is specific for NADH but can pass electrons to a variety of acceptors, including: phthalate oxygenase, cytochrome c, ferricyanide, and dichlorophenolindophenol. This system is similar to other bacterial oxygenase systems involved in aromatic degradation including: benzoate dioxygenase, toluene dioxygenase, benzene dioxygenase, and 4-methoxybenzoate demethoxylase. However, phthalate oxygenase can be isolated in large quantities and is more stable than most other such systems.
D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004591:Electrophoresis, Polyacrylamide Gel; D005486:Flavin Mononucleotide / Q000032:analysis; D007501:Iron / Q000032:analysis; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010088:Oxidoreductases / Q000302:isolation & purification*; D010105:Oxygenases / Q000302:isolation & purification*; D011549:Pseudomonas / Q000032:analysis*; D013053:Spectrophotometry; D013379:Substrate Specificity
D000596:Amino Acids; D009243:NAD; D005486:Flavin Mononucleotide; D007501:Iron; D010088:Oxidoreductases; C049135:phthalate oxygenase reductase; D010105:Oxygenases; C044732:phthalate dioxygenase