Purification and some properties of 2-halobenzoate 1,2-dioxygenase, a two-component enzyme system from Pseudomonas cepacia 2CBS.
The two components of the inducible 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS were purified to homogeneity. Yellow component B is a monomer (Mr, 37,500) with NADH-acceptor reductase activity. Ferricyanide, 2,6-dichlorophenol indophenol, and cytochrome c acted as electron acceptors. Component B was identified as an iron-sulfur flavoprotein containing 0.8 mol of flavin adenine dinucleotide, 1.7 mol of iron, and 1.7 mol of acid-labile sulfide per mol of enzyme. The isoelectric point was estimated to be pH 4.2. Component B was reduced by the addition of NADH. Red-brown component A (Mr, 200,000 to 220,000) is an iron-sulfur protein containing 5.8 mol of iron and 6.0 mol of acid-labile sulfide. The isoelectric point was within the range of pH 4.5 to 5.3. Component A could be reduced by dithionite or by NADH plus catalytic amounts of component B. Component A consisted of nonidentical subunits alpha (Mr, 52,000) and beta (Mr, 20,000). It contained approximately equimolar amounts of alpha and beta, and cross-linking studies suggested an alpha 3 beta 3 subunit structure of component A. The NADH- and Fe(2+)-dependent enzyme system was named 2-halobenzoate 1,2-dioxygenase, because it catalyzes the conversion of 2-fluoro-, 2-bromo-, 2-chloro-, and 2-iodobenzoate to catechol. 2-Halobenzoate 1,2-dioxygenase exhibited a very broad substrate specificity, but benzoate analogs with electron-withdrawing substituents at the ortho position were transformed preferentially.
Fetzner
S
S
Institut für Mikrobiologie Universität Hohenheim, Stuttgart, Germany.
Müller
R
R
Lingens
F
F
D000595:Amino Acid Sequence; D016956:Burkholderia cepacia / Q000201:enzymology*; D002723:Chlorobenzoates / Q000378:metabolism*; D003432:Cross-Linking Reagents; D005420:Flavoproteins / Q000737:chemistry / Q000302:isolation & purification; D006219:Halogens / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000302:isolation & purification; D007526:Isoelectric Point; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D010105:Oxygenases / Q000737:chemistry / Q000302:isolation & purification*; D013053:Spectrophotometry; D013379:Substrate Specificity
D002723:Chlorobenzoates; D003432:Cross-Linking Reagents; D005420:Flavoproteins; D006219:Halogens; D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D009243:NAD; C060415:2-chlorobenzoic acid; D010105:Oxygenases; C072183:2-chlorobenzoate 1,2-dioxygenase; D010100:Oxygen