Enhancement ofcis,cis-muconate productivity by overexpression of catechol 1,2-dioxygenase inPseudomonas putida BCM114

For enhancement ofcis,cis-muconate productivity from benzoate, catechol 1,2-dioxygenase (C12O) which catalyzes the rate-limiting step (catechol conversion tocis,cis-muconate) was cloned and expressed in recombinantPseudomonas putida BCM114. At higher benzoate concentrations (more than 15 mM),cis,cis-muconate productivity gradually decreased and unconverted catechol was accumulated up to 10 mM in the case of wildtypeP. putida BM014, whereascis,cis-muconate productivity continuously increased and catechol was completely transformed tocis,cis-muconate forP. putida BCM114. Specific C12O activity ofP. putida BCM114 was about three times higher than that ofP. putida BM014, and productivity was enhanced more than two times.     

Substrate Specificity of and Product Formation by Muconate Cycloisomerases: an Analysis of Wild-Type Enzymes and Engineered Variants

Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by converting cis,cis-muconate, the product of catechol ring cleavage, to (4S)-muconolactone. Chloromuconate cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the muconate cycloisomerases from Pseudomonas putida PRS2000 and Acinetobacter “calcoaceticus” ADP1. We show that they transform, in addition to cis,cis-muconate, 3-fluoro-, 2-methyl-, and 3-methyl-cis,cis-muconate with high specificity constants but not 2-fluoro-, 2-chloro-, 3-chloro-, or 2,4-dichloro-cis,cis-muconate. Based on known three-dimensional structures, variants of P. putida muconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids found in equivalent positions in chloromuconate cycloisomerases. Some of the variants had significantly increased specificity constants for 3-chloro- or 2,4-dichloromuconate (e.g., A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kinetic improvements were not accompanied by a change from protoanemonin to cis,cis-dienelactone as the product of 3-chloro-cis,cis-muconate conversion. The rate of 2-chloro-cis,cis-muconate turnover was not significantly improved, nor was this compound dehalogenated to any significant extent. However, the direction of 2-chloro-cis,cis-muconate cycloisomerization could be influenced by amino acid exchange. While the wild-type enzyme discriminated only slightly between the two possible cycloisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that can be changed independently of each other.     

Biodegradation of 3-nitrotoluene by Rhodococcus sp. strain ZWL3NT

A pure bacterial culture was isolated by its ability to utilize 3-nitrotoluene (3NT) as the sole source of carbon, nitrogen, and energy for growth. Analysis of its 16S rRNA gene showed that the organism (strain ZWL3NT) belongs to the genus Rhodococcus. A rapid disappearance of 3NT with concomitant release of nitrite was observed when strain ZWL3NT was grown on 3NT. The isolate also grew on 2-nitrotoluene, 3-methylcatechol and catechol. Two metabolites, 3-methylcatechol and 2-methyl-cis,cis-muconate, in the reaction mixture were detected after incubation of cells of strain ZWL3NT with 3NT. Enzyme assays showed the presence of both catechol 1,2-dioxygenase and catechol 2,3-dioxygenase in strain ZWL3NT. In addition, a catechol degradation gene cluster (catRABC cluster) for catechol ortho-cleavage pathway was cloned from this strain and cell extracts of Escherichia coli expressing CatA and CatB exhibited catechol 1,2-dioxygenase activity and cis,cis-muconate cycloisomerase activity, respectively. These experimental evidences suggest a novel pathway for 3NT degradation with 3-methylcatechol as a key metabolite by Rhodococcus sp. strain ZWL3NT.     

Differential DNA bending introduced by the Pseudomonas putida LysR-type regulator, CatR, at the plasmid-borne pheBA and chromosomal catBC promoters

The plasmid-borne pheBA operon of Pseudomonas putida strain PaW85 allows growth of the host cells on phenol. The promoter of this operon is activated by the chromosomally encoded LysR-type regulator CatR, in the presence of the inducer cis, cis-muconate. cis, cis -muconate is an intermediate of catechol degradation by the chromosomally encoded ortho or β-ketoadipate pathway. The catBC operon encodes two enzymes of the β-ketoadipate pathway and also requires CatR and cis, cis-muconate for its expression. The promoters of the pheBA and catBC operons are highly homologous, and since both respond to CatR, it is likely that the pheBA promoter was recruited from the ancestral catBC promoter. Gel shift assays and DNase I footprinting have shown that the pheBA promoter has a higher binding affinity for CatR than the catBC promoter. Like the catBC promoter, the pheBA promoter forms two complexes (C1 and C2) with CatR in the absence of cis, cis-muconate, but only forms a single complex (C2) in the presence of cis, cis-muconate. Like the catBC promoter CatR repression binding site (RBS) and activation binding site (ABS) arrangement, the pheBA promoter demonstrates the presence of a 26 bp segment highly homologous to the RBS that is protected by CatR from DNase I digestion in the absence of the inducer. An additional 16 bp sequence, similar to the catBC promoter ABS, is protected only when the inducer cis-cis-muconate is present. The binding of CatR in absence of cis, cis -muconate bends the catBC and pheBA promoter regions to significantly different degrees, but CatR binding in the presence of cis, cis-muconate results in a similar degree of DNA bending. The evolutionary implications of the interactions of CatR with these two promoters are discussed.     

固定化胞外邻苯二酚1，2－双加氧酶的研究

首次将胞外邻苯二酚1,2－双加氧酶固定化,并用于制备顺,顺—己二烯二酸.该固定化酶表观活力高,使用范围扩大,耐酸性及耐碱性都有显著提高,并且使用稳定性好,得到的产物浓度及纯度均较高,酶与产物容易分离,整个工艺简单、独特、新颖,有利于工业化应用.     

Molecular properties of cis,cis-muconate cycloisomerase from Pseudomonas putida

cis,cis-Muconate cycloisomerase (cis,cis-muconate lactonizing enzyme, EC 5.5.1.1.) was purified in crystalline form from Pseudomonas putida. Ultracentrifugation studies, as well as gel filtration chromatography and electrophoresis, indicate that the enzyme is an oligomeric protein of molecular weight 252,000 (s_20,w 12.20 × 10^?13 s), which is built of six homologous protomers of molecular weight 42,000. Studies of enzyme crystals and enzyme molecules in the electron microscope suggest that the cis,cis-muconate cycloisomerase is a hexamer in which the six protomers are arranged in a dihedral point-group symmetry 32 (D₃). Each protomer has a diameter of 42.5Åand six protomers are associated in a structure with a trigonal antiprismatic geometry (a hexamer D₃ octahedron). This model could account for the dimensions most frequently observed by negative staining of the enzyme in solution. A model for the three-dimensional structure of enzyme crystals in which each hexameric enzyme molecule is surrounded by eight neighbouring enzyme molecules, is described.     

Metabolism of acenaphthylene via 1,2-dihydroxynaphthalene and catechol by Stenotrophomonas sp. RMSK

Stenotrophomonas sp. RMSK capable of degrading acenaphthylene as a sole source of carbon and energy was isolated from coal sample. Metabolites produced were analyzed and characterized by TLC, HPLC and mass spectrometry. Identification of naphthalene-1,8-dicarboxylic acid, 1-naphthoic acid, 1,2-dihydroxynaphthalene, salicylate and detection of key enzymes namely 1,2-dihydroxynaphthalene dioxygenase, salicylaldehyde dehydrogenase and catechol-1,2-dioxygenase in the cell free extract suggest that acenaphthylene metabolized via 1,2-dihydroxynaphthalene, salicylate and catechol. The terminal metabolite, catechol was then metabolized by catechol-1,2-dioxygenase to cis,cis-muconic acid, ultimately forming TCA cycle intermediates. Based on these studies, the proposed metabolic pathway in strain RMSK is, acenaphthylene → naphthalene-1,8-dicarboxylic acid → 1-naphthoic acid → 1,2-dihydroxynaphthalene → salicylic acid → catechol → cis,cis-muconic acid.     

Catabolism of 3,5-dichlorosalicylate by Pseudomonas species strain JWS

Abstract A Pseudomonas sp. strain JWS was isolated from an enrichment culture with 3,5-dichlorosalicylate as the sole source of carbon and energy. Additionally, 3-chloro-, 5-chloro-, and 3,5-dibromosalicylate, but not 4-chlorosalicylate were mineralized by the organism. During growth on the chlorosalicylates, stoichiometric amounts of chloride were released into the culture medium. In the presence of both salicylate and 3,5-dichlorosalicylate, high activities were induced for the turnover of non-halogenated as well as halogenated salicylates. Enzyme activities assayed in crude cell extracts which are responsible for the oxidation of catechol and its halogenated derivatives as well as those for cycloisomerization of cis,cis -muconate and its 2,4-dichloro derivative provided indications for the involvement of inducible type II catechol 1,2-dioxygenase and muconate cycloisomerase in biodegradation of halogenated salicylates.     

Substrate specificities of the chloromuconate cycloisomerases from Pseudomonas sp. B13, Ralstonia eutropha JMP134 and Pseudomonas sp. P51

The chloromuconate cycloisomerase of Pseudomonas sp. B13 was purified from 3-chlorobenzoate-grown wild-type cells while the chloromuconate cycloisomerases of Ralstonia eutropha JMP134 (pJP4) and Pseudomonas sp. P51 (pP51) were purified from Escherichia coli strains expressing the corresponding gene. Kinetic studies were performed with various chloro-, fluoro-, and methylsubstituted cis,cis-muconates. 2,4-Dichloro-cis,cis-muconate proved to be the best substrate for all three chloromuconate cycloisomerases. Of the three enzymes, TfdD of Ralstonia eutropha JMP134 (pJP4) was most specific, since its specificity constant for 2,4-dichloro-cis,cis-muconate was the highest, while the constants for cis,cis-muconate, 2-chloro- and 2,5-dichloro-cis,cis-muconate were especially poor. The sequence of ClcB of the 3-chlorobenzoate-utilizing strain Pseudomonas sp. B13 was determined and turned out to be identical to that of the corresponding enzyme of pAC27 (though slightly different from the published sequences). Corresponding to 2-chloro-cis,cis-muconate being a major metabolite of 3-chlorobenzoate degradation, the k _cat/K _m with 2-chloro-cis,cis-muconate was relatively high, while that with the still preferred substrate 2,4-dichloro-cis,cis-muconate was relatively low. This enzyme was thus the least specific and the least active among the three compared enzymes. TcbD of Pseudomonas sp. P51 (pP51) took an intermediate position with respect to both the degree of specificity and the activity with the preferred substrate. Received: 7 August 1998 / Received revision: 24 November 1998 / Accepted: 29 November 1998     

Metabolization of 3,5-dichlorocatechol by Alcaligenes eutrophus JMP 134

2,4-Dichloro-cis,cis-muconate is established as ringcleavage product in the degradation of 3,5-dichlorocatechol by Alcaligenes eutrophus JMP 134. The formerly described isomerization of 2-chloro-trans- to 2-chlorocis-4-carboxymethylenebut-2-en-4-olide as an essential catabolic step could not be certified.     

Metabolism of Aniline by Rhodococcus erythropolis AN–13

The metabolic pathway of aniline was examined in Rhodococcus erythropolis AN-13 that was isolated from soil when aniline was provided as a sole source of carbon and nitrogen. cis, cis-Muconic acid and β-ketoadipic acid were detected by thin-layer chromatography in an incubation mixture containing aniline and resting cells of this strain. These two carboxylic acids were also formed from catechol, when the substrate was incubated with cell-free extract of aniline-grown cells, and characterized spectrally as crystalline samples. Ammonia was released from aniline by resting cells. The cell-free extract of aniline-grown cells had a strong catechol 1,2-dioxygenase activity. Catechol, once formed from aniline, was apparently converted so rapidly to cis, cis-muconic acid that it could not be isolated. These results suggest that R. erythropolis AN-13 converted aniline to catechol with the release of ammonia and then mineralized catechol ultimately to inorganic end products, H₂O and CO₂, through the β ketoadipic acid pathway.     

Catabolism of biphenyl by Pseudomonas sp. NCIB 10643 and Nocardia sp. NCIB 10503

Summary The metabolism of biphenyl by Pseudomonas sp. NCIB 10643 is reported in detail; that of Nocardia sp. NCIB 10503 is briefly investigated. Both organisms dissimilate biphenyl by the same route via oxidation to 2,3-dihydroxybiphenyl, meta cleavage to a product identified as 2-hydroxy-6-oxo-phenylhexa-2,4-dienoate which is then cleaved to give benzoate. Benzoate is a deadend metabolite in the pseudomonad but in the nocardia is further catabolised to catechol and thence to cis, cis-muconate. The enzymes involved in the individual steps of the proposed pathway have been assayed. The proposed pathway differs from that previously suggested for Pseudomonas sp. NCIB 10643 but is the same as found in other pseudomonads. This is the first report of catabolism of biphenyl in an actinomycete.     

Accumulation of 2-chloromuconate during metabolism of 3-chlorobenzoate by Alcaligenes eutrophus JMP134

Alcaligenes eutrophus JMP134 metabolizes 3-chlorobenzoate via 3- (3CC) and 4-chlorocatechol (4CC) as central metabolites. Whereas 4CC was efficiently degraded without a build-up of significant quantities of intermediates, substantial amounts of 2-chloro-cis,cis-muconate (2CM) formed from 3CC were excreted as a result of the poor activity of dichloromuconate cycloisomerase for this compound. This pathway bottleneck can, using appropriate fermentation conditions, be exploited in the production of 2CM. Correspondence to: D. H. Pieper     

QM/MM study of the reaction mechanism of Cl-cis,cis-muconate with muconate lactonizing enzyme

The lactonization process of Cl-cis,cis-muconate catalyzed by anti-muconate lactonizing enzyme (anti-MLE) was studied theoretically with the aid of a combined quantum mechanics/molecular mechanics (QM/MM) approach. Two elementary processes steps involved in the lactanization process were investigated. The calculated energy barriers agree well with the experimental values. The present work provided the explicit structures of the enolate anion intermediates. The electrostatic influence analysis highlighted residues Arg51, Gln294 and TIP383 for the MLE-Cl-2 system and the residue Asn193 for the MLE-Cl-4 system as the possible mutation targets for rational design of anti-MLE in future enzyme modification.     

Purification and characterization of a novel type of protocatechuate 3,4-dioxygenase with the ability to oxidize 4-sulfocatechol

4-Aminobenzenesulfonate is degraded via 4-sulfocatechol by a mixed bacterial culture that consists of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. From the 4-sulfocatechol-degrading organism A. radiobacter strain S2, a dioxygenase that converted 4-sulfocatechol to 3-sulfomuconate was purified to homogeneity. The purified enzyme also converted protocatechuate with a similar catalytic activity to 3-carboxy-cis,cis-muconate. Furthermore, the purified enzyme oxidized 3,4-dihydroxyphenylacetate, 3,4-dihydroxycinnamate, catechol, and 3- and 4-methylcatechol. The enzyme had a mol. wt. of about 97,400 as determined by gel filtration and consisted of two different types of subunits with mol. wt. of about 23,000 and 28,500. The NH₂-terminal amino acid sequences of the two subunits were determined. An isofunctional dioxygenase was partially purified from H. palleronii strain S1. A. radiobacter strain S2 also induced, after growth with 4-sulfocatechol, an „ordinary“ protocatechuate 3,4-dioxygenase that did not oxidize 4-sulfocatechol. This enzyme was also purified to homogeneity, and its catalytic and structural characteristics were compared to the „4-sulfocatechol-dioxygenase“ from the same strain. Received: 5 February 1996 / Accepted: 18 April 1996     

Short communication: Benzene metabolism via the intradiol cleavage in a Rhodococcus sp.

Benzene was metabolized by Rhodococcus sp. 33 through the intradiol cleavage (ortho-) pathway producing cis-benzene glycol, catechol and cis, cis-muconic acid as the intermediates. This is the first elucidation of the pathway by which benzene is degraded by a gram-positive organism. The enzyme assays have also suggested that Rhodococcus 33 does not have a fully functional tricarboxylic acid cycle but may have an operational glyoxylate bypass.     

Biodegradation of 3-chlorobenzoate by Pseudomonas putida 10.2

Pseudomonas putida 10.2, a 3-chlorobenzoate (3CBa)-degrading bacterium, was isolated from a soil sample obtained from an agricultural area in Chiang Mai, Thailand. This bacterium could degrade 2mm 3CBa very rapidly with the concomitant formation of chloride ion when grown in mineral salt-yeast extract medium. The presence of glucose, lactose and pyruvate in the medium reduced the capability of this bacterium to degrade 3CBa. Metabolites such as 3-chlorocatechol (3CC), catechol and cis,cis-muconic acid (muconate) could be detected in the growth medium or in cell suspensions when 3CBa was used as the substrate. Furthermore, when crude enzyme extract prepared from 3CBa-grown P. putida 10.2 was incubated with 3CC, catechol and muconate could be detected in the reaction mixtures. Thus, the biodegradation pathway of 3CBa by P. putida 10.2 was proposed to involve transformation of 3CBa to 3CC. The dehalogenation step is believed to involve removal of chloride from 3CC to form catechol, which is subsequently converted to muconate.     

Immunological comparison of enzymes of the β-ketoadipate pathway

-Carboxy-cis,cis-muconate lactonizing enzyme and -carboxymuconolactone decarboxylase catalyze sequential reactions in the -ketoadipate pathway, the subunit sizes of the enzymes from Pseudomonas putida, biotype A, are 40000 and 13000, respectively. The cross reaction of antisera prepared against the enzymes was tested with the isofunctional enzymes formed by representatives of other bacterial species. Despite the differences in the subunit sizes of the enzymes, the antisera revealed the same general pattern: cross reaction was observed with the corresponding enzymes formed by other strains in the fluorescent Pseudomonas RNA homology group I and generally was not observed with enzymes from other Pseudomonas species or from other bacterial genera. Exceptions were provided by representatives of Pseudomonas cepacia. Members of this species are classified outside the fluorescent Pseudomonas RNA homology group. Nevertheless, the -carboxymuconolactone decarboxylases from these organisms formed precipitin bands with antisera prepared against the corresponding enzyme from P. putida, biotype A; the lactonizing enzymes from the two species did not appear to cross react. Immunodiffusion experiments with -carboxymuconolactone decarboxylase indicated that a common set of antigenic determinants for the enzyme is conserved among strains that have been classified together by other criteria; the relative immunological distances of the decarboxylases of each taxon from the reference P. putida, biotype A, enzyme were indicated by spurring patterns on Ouchterlony plates. These results suggested that the interspecific transfer of the structural gene for the enzyme is not a common event in Pseudomonas.Non-Standard Abbreviations CMLE -carboxy-cis,cis-muconate lactonizing enzyme (EC 5.5.1.2) - CMD -carboxymuconolactone decarboxylase (EC 4.1.1.44) - MLE cis,cis-muconate lactonizing enzyme (EC 5.5.1.1) - MI muconolactone isomerase (EC 5.3.3.4) Dedicated with affection and admiration to Professor R. Y. Stanier on his 60th birthday     

Degradation of p-nitrophenol by the phototrophic bacterium Rhodobacter capsulatus

The phototrophic bacterium Rhodobacter capsulatus detoxified p-nitrophenol and 4-nitrocatechol. The bacterium tolerated moderate concentrations of p-nitrophenol (up to 0.5 mM) and degraded it under light at an optimal O₂ pressure of 20 kPa. The bacterium did not metabolize the xenobiotic in the dark or under strictly anoxic conditions or high O₂ pressure. Bacterial growth with acetate in the presence of p-nitrophenol took place with the simultaneous release of nonstoichiometric amounts of 4-nitrocatechol, which can also be degraded by the bacterium. Crude extracts from R. capsulatus produced 4-nitrocatechol from p-nitrophenol upon the addition of NAD(P)H, although at a very low rate. A constitutive catechol 1,2-dioxygenase activity yielding cis,cis-muconate was also detected in crude extracts of R. capsulatus. Further degradation of 4-nitrocatechol included both nitrite- and CO₂-releasing steps since: (1) a strain of R. capsulatus (B10) unable to assimilate nitrate and nitrite released nitrite into the medium when grown with p-nitrophenol or 4-nitrocatechol, and the nitrite concentration was stoichiometric with the 4-nitrocatechol degraded, and (2) cultures of R. capsulatus growing microaerobically produced low amounts of ¹⁴CO₂ from radiolabeled p-nitrophenol. The radioactivity was also incorporated into cellular compounds from cells grown with uniformly labeled ¹⁴C-p-nitrophenol. From these results we concluded that the xenobiotic is used as a carbon source by R. capsulatus, but that only the strain able to assimilate nitrite (E1F1) can use p-nitrophenol as a nitrogen source. Received: 30 December 1996 / Accepted: 3 September 1997     

Methylcatechol 1,2-dioxygenase of <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> 6a is a new type of the catechol-cleaving enzyme

The strains Rhodococcus sp. 400, R. rhodochrous 172, and R. opacus 6a utilize 4-methylbenzoate as the only carbon and energy source. 4-Methylcatechol is a key intermediate of biodegradation. Its further conversion by all the strains proceeds via ortho-cleavage. The specific activity of catechol 1,2-dioxygenase assayed in crude extracts of Rhodococcus sp. 400 and R. rhodochrous 172 with 3- and 4-methylcatechols does not exceed the enzyme activity assayed with catechol. Two catechol 1,2-dioxygenases have been purified from the biomass of R. opacus strain 6a grown with 4-methylbenzoate. These enzymes differed in molecular mass and physicochemical and catalytic properties. One of these enzymes belongs to the type of enzymes cleaving the catechol ring and known as methylcatechol 1,2-dioxygenases. In bacteria of the Rhodococcus genus, such an enzyme is described here for the first time.