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

Biodegradation of halogenated organic compounds.

Publisher Summary The chapter introduces TOL plasmids and discusses pathways for toluene and xylenes catabolisms, regulation of catabolic genes, and evolution of catabolic pathways. The TOL pathway is one of three metabolic routes described by which toluene can be catabolized. The TOL plasmid encodes enzymes for conversion of the aromatic hydrocarbons to the corresponding carboxylic acids as well as the meta-pathway enzymes. The structure of the promoter sequences in pseudomonads involved in degradation of toluene to central metabolites encompasses four main elements. These are upper- and meta-pathway operon promoters and promoters of the two regulatory genes xylS and xy/R . The growth of TOL strains on benzoate (“Benzoate Curing”) is described. Uses of TOL plasmid genes in construction of novel strains and vectors are also discussed.

The TOL Plasmids: Determinants of the Catabolism of Toluene and the Xylenes

The taxonomic position of Pseudomonas sp. B13T, isolated as a 3-chlorobenzoate-degrading organism and used for several groundbreaking studies on the enzymology and genetics of the degradative pathway for haloaromatic compounds, was studied in detail. The previously performed physiological studies, the detection of ubiquinone Q-9, the polyamine pattern with putrescine and spermidine as major polyamines, a fatty acid profile with C18 : 1
ω7c, summed feature 3 and C16 : 0 as quantitatively the most important constituents and the 16S rRNA gene sequence demonstrated that Pseudomonas sp. B13T indeed belongs to the genus Pseudomonas. The sequence of the Pseudomonas sp. B13T 16S rRNA gene demonstrated a high degree of similarity with that of Pseudomonas citronellolis DSM 50332T (98.9 %), Pseudomonas nitroreducens DSM 14399T (98.7 %), Pseudomonas jinjuensis DSM 16612T (98.1 %) and Pseudomonas multiresinivorans DSM 17553T (98.7 %). Thus it was shown that strain Pseudomonas sp. B13T can be distinguished from related species by the ability/inability to assimilate N-acetylgalactosamine, d-galactose, putrescine, trans-aconitate and mesaconate and some differences in the fatty acid profile. The positioning of Pseudomonas sp. B13T as a separate taxon was finally verified by DNA hybridization, which demonstrated less than 45 % DNA–DNA similarity between strain Pseudomonas sp. B13T and the reference strains. On the basis of these results, Pseudomonas sp. B13T represents a novel species for which the name Pseudomonas knackmussii sp. nov. is proposed. The type strain is B13T (=DSM 6978T=LMG 23759T).

Pseudomonas knackmussii sp. nov.

Growth of Alcaligenes eutrophus JMP134 on 2,4-dichlorophenoxyacetate requires a 2,4-dichlorphenol hydroxylase encoded by gene tfdB. Catabolism of either 2,4-dichlorophenoxyacetate or 3-chlorobenzoate involves enzymes encoded by the chlorocatechol oxidative operon consisting of tfdCDEF, which converts 3-chloro- and 3,5-dichlorocatechol to maleylacetate and chloromaleylacetate, respectively. Transposon mutagenesis has localized tfdB and tfdCDEF to EcoRI fragment B of plasmid pJP4 (R. H. Don, A. J. Wieghtman, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 161:85-90, 1985). We present the complete nucleotide sequence of tfdB and tfdCDEF contained within a 7,954-base-pair HindIII-SstI fragment from EcoRI fragment B. Sequence and expression analysis of tfdB in Escherichia coli suggested that 2,4-dichlorophenol hydroxylase consists of a single subunit of 65 kilodaltons. The amino acid sequences of proteins encoded by tfdD and tfdE were found to be 63 and 53% identical to those of functionally similar enzymes encoded by clcB and clcD, respectively, from plasmid pAC27 of Pseudomonas putida. P. putida(pAC27) can utilize 3-chlorocatechol but not dichlorinated catechols. A region of DNA adjacent to clcD in pAC27 was found to be 47% identical in amino acid sequence to tfdF, a gene important in catabolizing dichlorocatechols. The region in pAC27 does not appear to encode a protein, suggesting that the absence of a functional trans-chlorodienelactone isomerase may prevent P. putida(pAC27) from utilizing 3,5-dichlorocatechol.

Organization and sequence analysis of the 2,4-dichlorophenol hydroxylase and dichlorocatechol oxidative operons of plasmid pJP4.

Derivatives of Pseudomonas sp. B13 which had acquired the capability to utilize 4-chloro- and 3,5-dichlorobenzoate as a consequence of the introduction of genes of the TOL plasmid of Pseudomonas putida mt-2 were studied. The utilization of these substrates, a property not shared by the parent strains, was shown to depend upon the combined activities of enzymes from the donor and from the recipient. During growth on 3-chloro-, 4-chloro-, and 3,5-dichlorobenzoate, predominantly the toluate 1,2-deoxygenase and both dihydrodihydroxybenzoate dehydrogenases of the parent strains were induced. On the other hand, no catechol 2,3-dioxygenase from P. putida mt-2 was detectable, so that degradation of chlorocatechols by the nonproductive meta-cleavage pathway was avoided. Instead of that, chlorocatechols were subject to ortho cleavage and totally degraded by the preexisting enzymes of Pseudomonas sp. B13.

W Reineke

Papers

Hybrid pathway for chlorobenzoate metabolism in Pseudomonas sp. B13 derivatives.