The inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-chloro- and 3-fluorocatechol and the iron-chelating agent Tiron (catechol-3,5-disulfonate) was studied. Whereas inactivation by Tiron is an oxygen-independent and mostly reversible process, inactivation by the 3-halocatechols was only observed in the presence of oxygen and was largely irreversible. The rate constants for inactivation (K2) were 1.62 × 10−3 sec−1 for 3-chlorocatechol and 2.38 × 10−3 sec−1 for 3-fluorocatechol. The inhibitor constants (Ki) were 23 μM for 3-chlorocatechol and 17 μM for 3-fluorocatechol. The kinetic data for 3-fluorocatechol could only be obtained in the presence of 2-mercaptoethanol. Besides inactivated enzyme, some 2-hydroxyhexa-2,4-diendioic acid was formed from 3-chlorocatechol, suggesting 5-chloroformyl-2-hydroxypenta-2,4-dienoic acid as the actual suicide product of meta-cleavage. A side product of 3-fluorocatechol cleavage is a yellow compound with the spectral characteristics of a 2-hydroxy-6-oxohexa-2,4-dienoic acid indicating 1,6-cleavage. Rates of inactivation by 3-fluorocatechol were reduced in the presence of superoxide dismutase, catalase, formate, and mannitol, which implies that superoxide anion, hydrogen peroxide, and hydroxyl radical exhibit additional inactivation.

Suicide Inactivation of Catechol 2,3-Dioxygenase from Pseudomonas putida mt-2 by 3-Halocatechols

Transposon Mutagenesis andCloning Analysis ofthePathways for Degradation of2,4-Dichlorophenoxyacetic Acidand 3-Chlorobenzoate inAlcaligenes eutrophus JMP134(pJP4)

Plasmid pJP4 permits its host bacterium, strain JMP134, to degrade and utilize as sole sources of carbon and energy 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid (R. H. Don and J. M. Pemberton, J. Bacteriol. 145:681-686, 1981). Mutagenesis of pJP4 by transposons Tn5 and Tn1771 enabled localization of five genes for enzymes involved in these catabolic pathways. Four of the genes, tfdB, tfdC, tfdD, and tfdE, encoded 2,4-dichlorophenol hydroxylase, dichlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, and chlorodienelactone hydrolase, respectively. No function has been assigned to the fifth gene, tfdF, although it may encode a trans-chlorodiene-lactone isomerase. Inactivation of genes tfdC, tfdD, and tfdE, which encode the transformation of dichlorocatechol to chloromaleylacetic acid, prevented host strain JMP134 from degrading both 3-chlorobenzoate and 2,4-dichlorophenoxyacetic acid, which indicates that the pathways for these two substrates utilize common enzymes for the dissimilation of chlorocatechols. Studies with cloned catabolic genes from pJP4 indicated that whereas all essential steps in the degradation of 2,4-dichlorophenoxyacetic acid are plasmid encoded, the conversion of 3-chlorobenzoate to chlorocatechol is specified by chromosomal genes.

Transposon mutagenesis and cloning analysis of the pathways for degradation of 2,4-dichlorophenoxyacetic acid and 3-chlorobenzoate in Alcaligenes eutrophus JMP134(pJP4).

Partially purified preparations of catechol 2,3-dioxygenase from toluene-grown cells of Pseudomonas putida catalyzed the stoichiometric oxidation of 3-methylcatechol to 2-hydroxy-6-oxohepta-2,4-dienoate. Other substrates oxidized by the enzyme preparation were catechol, 4-methylcatechol, and 4-fluorocatechol. The apparent Michaelis constants for 3-methylcatechol and catechol were 10.6 and 22.0 muM, respectively. Substitution at the 4-position decreases the affinity and activity of the enzyme for the substrate. Catechol 2,3-dioxygenase preparations did not oxidize 3-chlorocatechol. In addition, incubation of the enzyme with 3-chlorocatechol led to inactivation of the enzyme. Kinetic analyses revealed that both 3-chlorocatechol and 4-chlorocatechol were noncompetitive or mixed-type inhibitors of the enzyme. 3-Chlorocatechol (Ki = 0.14 muM) was a more potent inhibitor than 4-chlorocatechol (Ki = 50 muM). The effect of the ion-chelating agents Tiron and o-phenanthrolene were compared with that of 3-chlorocatechol on the inactivation of the enzyme. Each inhibitor appeared to remove iron from the enzyme, since inactive enzyme preparations could be fully reactivated by treatment with ferrous iron and a reducing agent.

Inhibition of catechol 2,3-dioxygenase from Pseudomonas putida by 3-chlorocatechol.

When I began this review my goal was to present a coherent overview of the biochemistry and regulation of the inducible P450 cytochromes of bacteria. Now, at the end, I wonder if a unified perspective is possible at this time. On the basis of admittedly limited data, bacterial P450 systems seem as different from each other as they are, as a group, from the mammalian P450 cytochromes. The most obvious physical difference between the bacterial monooxygenases and their mammalian counterparts is solubility; with several possible exceptions (69, 70, 76), bacterial P450s are soluble whereas the microsomal and mitochondrial P450s are membrane-associated proteins. In structure and organization, however, the few well-characterized prokaryotic P450-dependent systems vary widely. The three-component arrangement is probably most common but even here variation is apparent. The P450cam putidaredoxin reductase contains only FAD and is quite specific for NADH (35, 39); the P450meg megaredoxin reductase contains only FMN and is specific for NADPH (59, 60). Putitive two-component P450 systems in bacteria have not yet been adequately characterized but the P450oct and P450npd monooxygenases (69, 70, 93) could well be organized in this way. The catalytically self-sufficient P450BM-3 is currently the only single-component P450-dependent monooxygenase known but additional examples of this arrangement may well be found in other bacteria. Paradoxically, P450BM-3 is structurally much more analogous to liver microsomal P450 systems than to any other bacterial P450 monooxygenase characterized to date. Another generally recognized difference between prokaryotic and eukaryotic P450s pertains to function; most known bacterial P450-dependent systems initiate the oxidation of recalcitrant carbon compounds so that the hosts can utilize them as sole carbon sources for growth. Some lower eukaryotes [certain yeasts, for example (134)] also employ P450-dependent systems in this way but, among most fungi as well as in higher eukaryotes, P450 cytochromes are involved in specific pathways of sterol or other lipid syntheses or, as in the mammalian liver microsomal systems, in detoxification reactions.(ABSTRACT TRUNCATED AT 400 WORDS)

P450BM-3 and other Inducible Bacterial P450 Cytochromes: Biochemistry and Regulation

Several classes of mutants of Pseudomonas putida (JT810) defective in the utilization of p-cymene as sole carbon source have been isolated. Selective enrichment of the mutants and for strains putatively cured of a degradative plasmid was achieved by incubation of cells in minimal growth media containing p-cymene (or p-cumate) and various halogenated analogs of the growth substrates or pathway intermediates. Analogs which led to successful enrichments included: p-chlorotoluene, p-bromotoluene, alpha-chloro-p-xylene, and p-iodobenzoate. A mutant strain, PpJT811, constitutive for the p-cymene pathway gave significantly greater enrichments of defective mutants than the wild-type parent PpJT810 after incubation with the halogenated analogs. It is suggested that the defective mutants are enriched because of the genetic alterations they possess, which confer immunity to a lethal synthesis performed by transformation of the analogs in clones possessing an intact p-cymene pathway. A nomenclature for the genetic organization of p-cymene pathway is described.

G J Wigmore

Papers

p-Cymene pathway in Pseudomonas putida: selective enrichment of defective mutants by using halogenated substrate analogs.