Pseudomonas aeruginosa 142 uses a three-component ortho-halobenzoate 1,2-dioxygenase for metabolism of 2,4-dichloro- and 2-chlorobenzoate.
Cell extracts of Pseudomonas aeruginosa 142, which was previously isolated from a polychlorinated biphenyl-degrading consortium, were shown to degrade 2,4-dichlorobenzoate, 2-chlorobenzoate, and a variety of other substituted ortho-halobenzoates by a reaction that requires oxygen, NADH, Fe(II), and flavin adenine dinucleotide. By using extracts that were chromatographically depleted of chlorocatechol and catechol 1,2-dioxygenase activities, products of the initial reaction with 2,4- or 2,5-dichlorobenzoate and 2-chlorobenzoate were identified by mass spectrometry as 4-chlorocatechol and catechol. In contrast to the well-characterized benzoate dioxygenases or the recently described 2-halobenzoate 1,2-dioxygenase from P. cepacia 2CBS (S. Fetzner, R. Müller, and F. Lingens, J. Bacteriol. 174:279-290, 1992) that possess two protein components, the P. aeruginosa enzyme was resolved by ion-exchange chromatography into three components, each of which is required for activity. To verify the distinct nature of this enzyme, we purified, characterized, and identified one component as a ferredoxin (M(r), approximately 13,000) containing a single [2Fe-2S] Rieske-type cluster (electron paramagnetic resonance spectroscopic values of gx = 1.82, gy = 1.905, and gz = 2.02 in the reduced state) that is related in sequence to ferredoxins found in the naphthalene and biphenyl three-component dioxygenase systems. By analogy to these enzymes, we propose that the P. aeruginosa ferredoxin serves as an electron carrier between an NADH-dependent ferredoxin reductase and the terminal component of the ortho-halobenzoate 1,2-dioxygenase. The broad specificity and high regiospecificity of the enzyme make it a promising candidate for use in the degradation of mixtures of chlorobenzoates.
Romanov
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Institute for Biochemistry and Physiology of Microorganisms, Russian Academy of Science, Pushchino, Moscow Region.
Hausinger
R P
RP
D000595:Amino Acid Sequence; D002396:Catechols / Q000378:metabolism; D002723:Chlorobenzoates / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000378:metabolism*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D002396:Catechols; D002723:Chlorobenzoates; C070735:2,4-dichlorobenzoic acid; C060415:2-chlorobenzoic acid; D010105:Oxygenases; C087482:ortho-halobenzoate 1,2-dioxygenase; C038861:4-chlorocatechol; C034221:catechol