Of all NMR observable isotopes 19F is the one perhaps most convenient for studies on biodegradation of environmental pollutants. The reasons underlying this potential of 19F NMR are discussed and illustrated on the basis of a study on the biodegradation of fluorophenols by four Rhodococcus strains. The results indicate marked differences between the biodegradation pathways of fluorophenols among the various Rhodococcus species. This holds not only for the level and nature of the fluorinated biodegradation pathway intermediates that accumulate, but also for the regioselectivity of the initial hydroxylation step. Several of the Rhodococcus species contain a phenol hydroxylase that catalyses the oxidative defluorination of ortho-fluorinated di- and trifluorophenols. Furthermore, it is illustrated how the 19F NMR technique can be used as a tool in the process of identification of an accumulated unknown metabolite, in this case most likely 5-fluoromaleylacetate. Altogether, the 19F NMR technique proved valid to obtain detailed information on the microbial biodegradation pathways of fluorinated organics, but also to provide information on the specificity of enzymes generally considered unstable and, for this reason, not much studied so far.
The biochemical characterization of the muconate and the chloromuconate cycloisomerases of the chlorophenol-utilizing Rhodococcus erythropolis strain 1CP previously indicated that efficient chloromuconate conversion among the gram-positive bacteria might have evolved independently of that among gram-negative bacteria. Based on sequences of the N terminus and of tryptic peptides of the muconate cycloisomerase, a fragment of the corresponding gene has now been amplified and used as a probe for the cloning of catechol catabolic genes from R. erythropolis. The clone thus obtained expressed catechol 1,2-dioxygenase, muconate cycloisomerase, and muconolactone isomerase activities. Sequencing of the insert on the recombinant plasmid pRER1 revealed that the genes are transcribed in the order catA catB catC. Open reading frames downstream of catC may have a function in carbohydrate metabolism. The predicted protein sequence of the catechol 1,2-dioxygenase was identical to the one from Arthrobacter sp. strain mA3 in 59% of the positions. The chlorocatechol 1,2-dioxygenases and the chloromuconate cycloisomerases of gram-negative bacteria appear to be more closely related to the catechol 1,2-dioxygenases and muconate cycloisomerases of the gram-positive strains than to the corresponding enzymes of gram-negative bacteria.
Extracellular laccases from submerged cultures of Coriolus versicolor BKM F-116, Panus tigrinus 8/18, Phlebia radiata 79 (ATCC 64658), Phlebia tremellosa 77-51 and from cultures of Pa. tigrinus 8/18, Ph. radiata 79 and Agaricus bisporus D-649 grown on wheat straw (solid-state fermentation) were purified. All enzymes from submerged cultures had a blue colour and characteristic absorption and EPR spectra. Laccases from the solid-state cultures were yellow-brown and had no typical blue oxidase spectra and also showed atypical EPR spectra. Comparison of N-terminal amino acid sequences of purified laccases showed high homology between blue and yellow-brown laccase forms. Formation of yellow laccases as a result of binding of lignin-derived molecules by enzyme protein is proposed.
Muconate cycloisomerase (EC 5.5.1.1) and chloromuconate cycloisomerase (EC 5.5.1.7) were purified from extracts of Rhodococcus erythropolis 1CP cells grown with benzoate or 4-chlorophenol, respectively. Both enzymes discriminated between the two possible directions of 2-chloro-cis,cis-muconate cycloisomerization and converted this substrate to 5-chloromuconolactone as the only product. In contrast to chloromuconate cycloisomerases of gram-negative bacteria, the corresponding R. erythropolis enzyme is unable to catalyze elimination of chloride from (؉)-5-chloromuconolactone. Moreover, in being unable to convert (؉)-2-chloromuconolactone, the two cycloisomerases of R. erythropolis 1CP differ significantly from the known muconate and chloromuconate cycloisomerases of gram-negative strains. The catalytic properties indicate that efficient cycloisomerization of 3-chloro-and 2,4-dichloro-cis,cis-muconate might have evolved independently among gram-positive and gram-negative bacteria.Many chloroaromatic compounds are degraded by bacteria via chlorocatechols as central intermediates. Further catabolism involves ortho-cleavage of the chlorocatechols to chlorosubstituted cis,cis-muconates as well as cycloisomerization and dechlorination of the latter, yielding dienelactones (4-carboxymethylenebut-2-en-4-olides) which are hydrolyzed and finally funneled into the ubiquitous 3-oxoadipate pathway (Fig. 1). Despite much of the early work having been done with an Arthrobacter sp. (3,8,31) and despite many reports of transformation of halogenated aromatic compounds by gram-positive bacteria (recently reviewed in reference 35), the enzymology and genetics of the modified ortho-cleavage pathway outlined above have been elucidated almost exclusively in gram-negative strains. They usually contain separate sets of enzymes for catechol and chlorocatechol conversion, which differ from each other with respect to the affinities and turnover rates for chlorosubstituted catechols or the metabolites formed from them (6,21,25).The gram-positive strain Rhodococcus erythropolis 1CP has previously been reported to utilize 4-chlorophenol and 2,4-dichlorophenol as sole sources of carbon and energy (10). After some adaptation, it also grows slowly with 3-chlorophenol but not with 2-chlorophenol. Like many gram-negative strains, R. erythropolis 1CP possesses separate catechol and chlorocatechol catabolic enzymes (14, 16). The substrate preferences of the chlorocatechol 1,2-dioxygenase (15) and of the dienelactone hydrolase (16) of R. erythropolis 1CP suggest that, corresponding to the growth substrates, only a 4-chlorocatechol branch and a 3,5-dichlorocatechol branch are functional in strain 1CP, but there is no 3-chlorocatechol branch (Fig. 1). In this paper, we show that the substrate preference of the R. erythropolis 1CP chloromuconate cycloisomerase fits well with those of the dioxygenase and of the hydrolase. Moreover, 2-chloro-cis,cis-muconate was found to be converted to only one product, 5-chloromuconolactone, by both the muconate and the ...
The enzyme which cleaves the benzene ring of 6-chlorohydroxyquinol was purified to apparent homogeneity from an extract of 2,4,6-trichlorophenol-grown cells of Streptomyces rochei 303. Like the analogous enzyme from Azotobacter sp. strain GP1, it exhibited a highly restricted substrate specificity and was able to cleave only 6-chlorohydroxyquinol and hydroxyquinol and not catechol, chlorinated catechols, or pyrogallol. No extradiolcleaving activity was observed. In contrast to 6-chlorohydroxyquinol 1,2-dioxygenase from Azotobacter sp. strain GP1, the S. rochei enzyme had a distinct preference for 6-chlorohydroxyquinol over hydroxyquinol (k cat /K m ؍ 1.2 and 0.57 s ؊1 ⅐ M ؊1 , respectively). The enzyme from S. rochei appears to be a dimer of two identical 31-kDa subunits. It is a colored protein and was found to contain 1 mol of iron per mol of enzyme. The NH 2 -terminal amino acid sequences of 6-chlorohydroxyquinol 1,2-dioxygenase from S. rochei 303 and from Azotobacter sp. strain GP1 showed a high degree of similarity.Two pathways for the aerobic degradation of chlorophenols have been described so far: one via chlorocatechols and the other via chlorohydroquinones (2, 9, 10, 15). A predominant catabolic route for the compounds carrying one or two chlorine substituents was shown to be the modified ortho-cleavage pathway (14,18,24). In this pathway, chlorocatechols formed by introduction of a second hydroxy group are subjected to intradiol cleavage. In the case of chlorophenol degradation through the chlorohydroquinone pathway, the introduction of a third hydroxy group leads to the formation of hydroxyquinol or chlorohydroxyquinol (3, 13). Both unsubstituted and chlorinated 1,2,4-trihydroxybenzene were shown to be subject to ortho cleavage in Streptomyces rochei 303 (13). Extracts from cells grown in the presence of 2-chloro-or 2,4-dichlorophenol were found to prefer hydroxyquinol over 6-chlorohydroxyquinol as a ring fission substrate, whereas extracts from cells grown on 2,6-dichloro-or 2,4,6-trichlorophenol showed a preference for the chlorinated substrate (13). These results led us to suggest that two different dioxygenases play a role in the cleavage of the trihydroxylated aromatic ring: 6-chlorohydroxyquinol 1,2-dioxygenase in the ring fission of 6-chlorohydroxyquinol, the proposed intermediate of 2,4,6-trichloro-and 2,6-dichlorophenol degradation, and a second dioxygenase in the cleavage of hydroxyquinol, the proposed intermediate of 2,4-dichloro-and 2-chlorophenol degradation (Fig. 1) (13).In this paper, we describe the purification and characterization of 6-chlorohydroxyquinol 1,2-dioxygenase. Three other hydroxyquinol 1,2-dioxygenases have been purified and characterized so far (21,25,34). One of them takes part in the cleavage of hydroxyquinol, found as an intermediate of 4-hydroxybenzoate degradation in Trichosporon cutaneum (34).The second dioxygenase, from the basidiomycete Phanerochaete chrysosporium, catalyzes a key step in the degradation of vanillate, an intermediate in lignin degradation (25)....
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