Maleylacetate reductase of Pseudomonas sp. strain B13: dechlorination of chloromaleylacetates,metabolites in the degradation of chloroaromatic compounds

The maleylacetate reductase of 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 has been purified 50-fold. The enzyme converted 2-chloromaleylacetate to 3-oxoadipate with temporary occurrence of maleylacetate; 1 mol of chloride was eliminated during the conversion of 1 mol of 2-chloro- and 2,3-dichloromaleylacetate; 2 mol of NADH were consumed per mol of 2-chloro- and 2,3-dichloromaleylacetate while only 1 mol was necessary to catalyze the conversion of maleylacetate or 2-methylmaleylacetate. The maleylacetate reductase failed to use fumarylacetate as a substrate. The role of the enzyme in the chloroaromatics degradation is discussed.     

Purification of hydroxyquinol 1,2-dioxygenase and maleylacetate reductase: the lower pathway of 2,4,5-trichlorophenoxyacetic acid metabolism by Burkholderia cepacia AC1100.

The enzyme hydroxyquinol 1,2-dioxygenase, which catalyzes ortho cleavage of hydroxyquinol (1,2,4-trihydroxybenzene) to produce maleylacetate, was purified from Escherichia coli cells containing the tftH gene from Burkholderia cepacia AC1100. Reduction of the double bond in maleylacetate is catalyzed by the enzyme maleylacetate reductase, which was also purified from E. coli cells, these cells containing the tftE gene from B. cepacia AC1100. The two enzymes together catalyzed the conversion of hydroxyquinol to 3-oxoadipate. The purified hydroxyquinol 1,2-dioxygenase was specific for hydroxyquinol and was not able to use catechol, tetrahydroxybenzene, 6-chlorohydroxyquinol, or 5-chlorohydroxyquinol as its substrate. The native molecular mass of hydroxyquinol 1,2-dioxygenase was 68 kDa, and the subunit size of the protein was 36 kDa, suggesting a dimeric protein of identical subunits.     

Purification and characterization of maleylacetate reductase from Alcaligenes eutrophus JMP134(pJP4).

Maleylacetate reductase (EC 1.3.1.32) plays a major role in the degradation of chloroaromatic compounds by channeling maleylacetate and some of its substituted derivatives into the 3-oxoadipate pathway. The enzyme was purified to apparent homogeneity from an extract of 2,4-dichlorophenoxyacetate (2,4-D)-grown cells of Alcaligenes eutrophus JMP134. Maleylacetate reductase appears to be a dimer of two identical subunits of 35 kDa. The pI was determined to be at pH 5.4. There was no indication of a flavin prosthetic group. The enzyme was inactivated by p-chloromercuribenzoate but not by EDTA, 1,10-phenanthroline, or dithiothreitol. Maleylacetate and 2-chloromaleylacetate were converted with similar efficiencies (with NADH as cosubstrate, Km = 31 microM for each substrate and kcat = 8,785 and 7,280/min, respectively). NADH was preferred to NADPH as the cosubstrate. Upon reduction of 2-chloramaleylacetate by the purified enzyme, chloride was liberated and the resulting maleylacetate was further reduced by a second NADH. These results and the kinetic parameters suggest that the maleylacetate reductase is sufficient to channel the 2,4-D degradation intermediate 2-chloromaleylacetate into the 3-oxoadipate pathway. In a data base search the NH2-terminal sequence of maleylacetate reductase was found to be most similar to that of TfdF, a pJP4-encoded protein of as-yet-unknown function in 2,4-D degradation.     

Evidence that operons tcb, tfd, and clc encode maleylacetate reductase, the fourth enzyme of the modified ortho pathway.

The maleylacetate reductase from Pseudomonas sp. strain B13 functioning in the modified ortho pathway was purified and digested with trypsin. The polypeptides separated by high-performance liquid chromatography were sequenced. Alignments with the polypeptides predicted from the tfdF and tcbF genes located on plasmids pJP4 of the 2,4-dichlorophenoxyacetate-degrading Alcaligenes eutrophus JMP134 and pP51 of the 1,2,4-trichlorobenzene-degrading Pseudomonas sp. strain P51 as well as polypeptides predicted from the tftE gene located on the chromosome of the 2,4,5-trichlorophenoxyacetate-degrading Burkholderia cepacia AC1100 were obtained. In addition, the deduced protein sequence encoded by the nucleotide sequence downstream of clcD on plasmid pAC27 of the 3-chlorobenzoate-degrading Pseudomonas putida AC866 was tested for homology. Significant sequence similarities with the polypeptides encoded by the tfdF, tcbF, and tftE genes as well as the nucleotide sequence downstream of the clcD gene gave evidence that these genes might encode maleylacetate reductases. A NAD-binding motif in a beta alpha beta-element was detected.     

Novel Pathway for Conversion of Chlorohydroxyquinol to Maleylacetate in Burkholderia cepacia AC1100

Burkholderia cepacia AC1100 metabolizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) via formation of 5-chlorohydroxyquinol (5-CHQ), hydroxyquinol (HQ), maleylacetate, and β-oxoadipate. The step(s) leading to the dechlorination of 5-CHQ to HQ has remained unidentified. We demonstrate that a dechlorinating enzyme, TftG, catalyzes the conversion of 5-CHQ to hydroxybenzoquinone, which is then reduced to HQ by a hydroxybenzoquinone reductase (HBQ reductase). HQ is subsequently converted to maleylacetate by hydroxyquinol 1,2-dioxygenase (HQDO). All three enzymes were purified. We demonstrate specific product formation by colorimetric assay and mass spectrometry when 5-CHQ is treated successively with the three enzymes: TftG, TftG plus HBQ reductase, and TftG plus HBQ reductase plus HQDO. This study delineates the complete enzymatic pathway for the degradation of 5-CHQ to maleylacetate.     

Purification and characterization of maleylacetate reductase from Nocardioides simplex 3E utilizing phenoxyalcanoic herbicides 2,4-D and 2,4,5-T.

Maleylacetate reductase was isolated and purified from the Gram-positive strain Nocardioides simplex 3E which is able to utilize the phenoxyalcanoic herbicides 2,4-D and 2,4,5-T. Cells were grown on 2,4-D as the sole carbon source. The enzyme was purified by 380-fold with 3.0% yield. The purified maleylacetate reductase is a homodimer with subunit molecular mass of 37 kD. The enzyme required NADH as a cofactor; the Km for maleylacetate is 25 microM; Vmax (with NADH as cofactor) and kcat are 185 U/mg and 6845 min-1, respectively. The enzyme is very unstable; its pH and temperature optima are at 7.0-7.1 and 50 degrees C, respectively.     

New bacterial pathway for 4- and 5-chlorosalicylate degradation via 4-chlorocatechol and maleylacetate in Pseudomonas sp. strain MT1

Pseudomonas sp. strain MT1 is capable of degrading 4- and 5-chlorosalicylates via 4-chlorocatechol, 3-chloromuconate, and maleylacetate by a novel pathway. 3-Chloromuconate is transformed by muconate cycloisomerase of MT1 into protoanemonin, a dominant reaction product, as previously shown for other muconate cycloisomerases. However, kinetic data indicate that the muconate cycloisomerase of MT1 is specialized for 3-chloromuconate conversion and is not able to form cis-dienelactone. Protoanemonin is obviously a dead-end product of the pathway. A trans-dienelactone hydrolase (trans-DLH) was induced during growth on chlorosalicylates. Even though the purified enzyme did not act on either 3-chloromuconate or protoanemonin, the presence of muconate cylcoisomerase and trans-DLH together resulted in considerably lower protoanemonin concentrations but larger amounts of maleylacetate formed from 3-chloromuconate than the presence of muconate cycloisomerase alone resulted in. As trans-DLH also acts on 4-fluoromuconolactone, forming maleylacetate, we suggest that this enzyme acts on 4-chloromuconolactone as an intermediate in the muconate cycloisomerase-catalyzed transformation of 3-chloromuconate, thus preventing protoanemonin formation and favoring maleylacetate formation. The maleylacetate formed in this way is reduced by maleylacetate reductase. Chlorosalicylate degradation in MT1 thus occurs by a new pathway consisting of a patchwork of reactions catalyzed by enzymes from the 3-oxoadipate pathway (catechol 1,2-dioxygenase, muconate cycloisomerase) and the chlorocatechol pathway (maleylacetate reductase) and a trans-DLH.     

The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP‐10005 provides insights into the reaction mechanism of enzymes in its original family

Maleylacetate reductase plays a crucial role in catabolism of resorcinol by catalyzing the NAD(P)H‐dependent reduction of maleylacetate, at a carbon–carbon double bond, to 3‐oxoadipate. The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP‐10005, GraC, has been elucidated by the X‐ray diffraction method at 1.5 Å resolution. GraC is a homodimer, and each subunit consists of two domains: an N‐terminal NADH‐binding domain adopting an α/β structure and a C‐terminal functional domain adopting an α‐helical structure. Such structural features show similarity to those of the two existing families of enzymes in dehydroquinate synthase‐like superfamily. However, GraC is distinct in dimer formation and activity expression mechanism from the families of enzymes. Two subunits in GraC have different structures from each other in the present crystal. One subunit has several ligands mimicking NADH and the substrate in the cleft and adopts a closed domain arrangement. In contrast, the other subunit does not contain any ligand causing structural changes and adopts an open domain arrangement. The structure of GraC reveals those of maleylacetate reductase both in the coenzyme, substrate‐binding state and in the ligand‐free state. The comparison of both subunit structures reveals a conformational change of the Tyr326 loop for interaction with His243 on ligand binding. Structures of related enzymes suggest that His243 is likely a catalytic residue of GraC. Mutational analyses of His243 and Tyr326 support the catalytic roles proposed from structural information. The crystal structure of GraC characterizes the maleylacetate reductase family as a third family in the dehydroquinate synthase‐like superfamily. Proteins 2016; 84:1029–1042. © 2016 Wiley Periodicals, Inc.     

Pseudomonas aeruginosa strain RW41 mineralizes 4-chlorobenzenesulfonate, the major polar by-product from DDT manufacturing

Pseudomonas aeruginosa RW41 is the first bacterial strain, which could be isolated by virtue of its capability to mineralize 4-chlorobenzenesulfonic acid (4CBSA), the major polar by-product of the chemical synthesis of 1,1,1-trichloro-2,2-bis-(4-chlorophenyl)ethane (DDT). This capability makes the isolate a promising candidate for the development of bioremediation technologies. The bacterial mineralization of 4CBSA proceeds under oxygenolytic desulfonation and transient accumulation of sulfite which then is oxidized to sulfate. High enzyme activities for the turnover of 4-chlorocatechol were measured. The further catabolism proceeded through 3-chloromuconate and, probably, the instable 4-chloromuconolactone, which is directly hydrolyzed to maleylacetate. Detectable levels of maleylacetate reductase were only present when cells were grown with 4CBSA. When the ordinary catechol pathway was induced during growth on benzenesulfonate, catechol was ortho-cleaved to cis,cis-muconate and a partially purified muconate cycloisomerase transformed it to muconolactone in vitro. The same enzyme transformed 3-chloro-cis,cis-muconate into cis-dienelactone (76%) and the antibiotically active protoanemonin (24%). These observations are indicative for a not yet highly evolved catabolism for halogenated substrates by bacterial isolates from environmental samples which, on the other hand, are able to productively recycle sulfur and chloride ions from synthetic haloorganosulfonates.     

Degradation of chloroaromatics: purification and characterization of maleylacetate reductase from Pseudomonas sp. strain B13.

Maleylacetate reductase of Pseudomonas sp. strain B13 was purified to homogeneity by chromatography on DEAE-cellulose, Butyl-Sepharose, Blue-Sepharose, and Sephacryl S100. The final preparation gave a single band by polyacrylamide gel electrophoresis under denaturing conditions and a single symmetrical peak by gel filtration under nondenaturing conditions. The subunit M(r) value was 37,000 (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Estimation of the native M(r) value by gel filtration gave a value of 74,000 with a Superose 6 column, indicating that the enzyme is dimeric. The pH and temperature optima were 5.4 and 50 degrees C, respectively. The pI of the enzyme was estimated to be 7.0. The apparent Km values for maleylacetate and NADH were 58 and 30 microM, respectively, and the maximum velocity was 832 U/mg of protein for maleylacetate. Maleylacetate and various substituted maleylacetates, such as 2-chloro- and 2-methyl-maleylacetate, were reduced at significant rates. NADPH was also used as a cofactor instead of NADH with nearly the same Vmax value, but its Km value was estimated to be 77 microM. Reductase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group in the enzyme.     

Cloning,Overexpression, Purification,and Characterization of the Maleylacetate Reductase from <Emphasis Type="Italic">Sphingobium chlorophenolicum</Emphasis> Strain ATCC 53874

The final enzyme in the pentachlorophenol (PCP) biodegradation pathway in Sphingobium chlorophenolicum is maleylacetate reductase (PcpE), which catalyzes the reductive dehalogenation of 2-chloromaleylacetate to maleylacetate and the subsequent reduction of malyelacetate to 3-oxoadipate. In this study, the pcpE gene was cloned from S. chlorophenolicum strain ATCC 53874 and overexpressed in Escherichia coli BL21-AI cells. The recombinant PcpE, purified to higher than 95% purity using affinity chromatography, exhibited optimal activity at pH 7.0. The kinetic parameters k _cat and K _m were 1.2 ± 0.3 s⁻¹ and 0.09 ± 0.04 mM, respectively, against maleylacetate under the optimal pH. In addition, the purified PcpE was able to restore PCP-degrading capability to S. chlorophenolicum strain ATCC 39723, implicating that there was no functional PcpE in the ATCC 39723 strain.     

Monitoring key reactions in degradation of chloroaromatics by in situ (1)H nuclear magnetic resonance: solution structures of metabolites formed from cis-dienelactone

A (1)H nuclear magnetic resonance ((1)H NMR) assay was used to study the enzymatic transformation of cis-dienelactone, a central intermediate in the degradation of chloroaromatics. It was shown that the product of the cis-dienelactone hydrolase reaction is maleylacetate, in which there is no evidence for the formation of 3-hydroxymuconate. Under acidic conditions, the product structure was 4-carboxymethyl-4-hydroxybut-2-en-4-olide. Maleylacetate was transformed by maleylacetate reductase into 3-oxoadipate, a reaction competing with spontaneous decarboxylation into cis-acetylacrylate. One-dimensional (1)H NMR in (1)H(2)O could thus be shown to be an excellent noninvasive tool for monitoring enzyme activities and assessing the solution structure of substrates and products.     

Metabolism of resorcinylic compounds by bacteria: alternative pathways for resorcinol catabolism in Pseudomonas putida.

Two strains of Pseudomonas putida isolated by enrichment cultures with orcinol as the sole source of carbon were both found to grow with resorcinol. Data are presented which show that one strain (ORC) catabolizes resorcinol by a metabolic pathway, genetically and mechanistically distinct from the orcinol pathway, via hydroxyquinol and ortho oxygenative cleavage to give maleylacetate, but that the other strain (O1) yields mutants that utilize resorcinol. One mutant strain, designated O1OC, was shown to be constitutive for the enzymes of the orcinol pathway. After growth of this strain on resorcinol, two enzymes of the resorcinol pathway are also induced, namely hydroxyquinol 1,2-oxygenase and maleylacetate reductase. Thus hydroxyquniol, formed from resorcinol, undergoes both ortho and meta diol cleavage reactions with the subsequent formation of both pyruvate and maleylacetate. Evidence was not obtained for the expression of resorcinol hydroxylase in strain O1OC; the activity of orcinol hydroxylase appears to be recruited for this hydroxylation reaction. P. putida ORC, on the other hand, possesses individual hydroxylases for orcinol and resorcinol, which are specifically induced by growth on their respective substrates. The spectral changes associated with the enzymic and nonenzymic oxidation of hydroxyquinol are described. Maleylacetate was identified as the product of hydroxyquinol oxidation by partially purified extracts obtained from P. putida ORC grown with resorcinol. Its further metabolism was reduced nicotinamide adenine dinucleotide dependent.     

Purification, molecular cloning, and catalytic activity of Schizosaccharomyces pombe pyridoxal reductase. A possible additional family in the aldo-keto reductase superfamily.

Pyridoxal reductase (PL reductase), which catalyzes reduction of PL by NADPH to form pyridoxine and NADP(+), was purified from Schizosaccharomyces pombe. The purified enzyme was very unstable but was stabilized by low concentrations of various detergents such as Tween 40. The enzyme was a monomeric protein with the native molecular weight of 41,000 +/- 1,600. The enzyme showed a single absorption peak at 280 nm (E(1%) = 10.0). PL and 2-nitrobenzaldehyde were excellent substrates, and no measurable activity was observed with short chain aliphatic aldehydes; substrate specificity of PL reductase was obviously different from those of yeast aldo-keto reductases (AKRs) so far purified. The peptide sequences of PL reductase were identical with those in a hypothetical 333-amino acid protein from S. pombe (the DDBJ/EMBL/GenBank(TM) accession number D89205). The gene corresponding to this protein was expressed in Escherichia coli, and the purified protein was found to have PL reductase activity. The recombinant PL reductase showed the same properties as those of native PL reductase. PL reductase showed only low sequence identities with members of AKR superfamily established to date; it shows the highest identity (18.5%) with human Shaker-related voltage-gated K(+) channel beta2 subunit. The elements of secondary structure of PL reductase, however, distributed similarly to those demonstrated in the three-dimensional structure of human aldose reductase except that loop A region is lost, and loop B region is extended. Amino acid residues involved in substrate binding or catalysis are also conserved. Conservation of these features, together with the major modifications, establish PL reductase as the first member of a new AKR family, AKR8.     

Elucidation of the 4-hydroxyacetophenone catabolic pathway in Pseudomonas fluorescens ACB

The catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB is known to proceed through the intermediate formation of hydroquinone. Here, we provide evidence that hydroquinone is further degraded through 4-hydroxymuconic semialdehyde and maleylacetate to beta-ketoadipate. The P. fluorescens ACB genes involved in 4-hydroxyacetophenone utilization were cloned and characterized. Sequence analysis of a 15-kb DNA fragment showed the presence of 14 open reading frames containing a gene cluster (hapCDEFGHIBA) of which at least four encoded enzymes are involved in 4-hydroxyacetophenone degradation: 4-hydroxyacetophenone monooxygenase (hapA), 4-hydroxyphenyl acetate hydrolase (hapB), 4-hydroxymuconic semialdehyde dehydrogenase (hapE), and maleylacetate reductase (hapF). In between hapF and hapB, three genes encoding a putative intradiol dioxygenase (hapG), a protein of the Yci1 family (hapH), and a [2Fe-2S] ferredoxin (hapI) were found. Downstream of the hap genes, five open reading frames are situated encoding three putative regulatory proteins (orf10, orf12, and orf13) and two proteins possibly involved in a membrane efflux pump (orf11 and orf14). Upstream of hapE, two genes (hapC and hapD) were present that showed weak similarity with several iron(II)-dependent extradiol dioxygenases. Based on these findings and additional biochemical evidence, it is proposed that the hapC and hapD gene products are involved in the ring cleavage of hydroquinone.     

Recruitment of a chromosomally encoded maleylacetate reductase for degradation of 2,4-dichlorophenoxyacetic acid by plasmid pJP4.

When Pseudomonas aeruginosa PAO1c or P. putida PPO200 or PPO300 carry plasmid pJP4, which encodes enzymes for the degradation of 2,4-dichlorophenoxyacetic acid (TFD) to 2-chloromaleylacetate, cells do not grow on TFD and UV-absorbing material with spectral characteristics of chloromaleylacetate accumulates in the culture medium. Using plasmid pRO1727, we cloned from the chromosome of a nonfluorescent pseudomonad, Pseudomonas sp. strain PKO1, 6- and 0.5-kilobase BamHI DNA fragments which contain the gene for maleylacetate reductase. When carrying either of the recombinant plasmids, pRO1944 or pRO1945, together with pJP4, cells of P. aeruginosa or P. putida were able to utilize TFD as a sole carbon source for growth. A novel polypeptide with an estimated molecular weight of 18,000 was detected in cell extracts of P. aeruginosa carrying either plasmid pRO1944 or plasmid pRO1945. Maleylacetate reductase activity was induced in cells of P. aeruginosa or P. putida carrying plasmid pRO1945, as well as in cells of Pseudomonas strain PKO1, when grown on L-tyrosine, suggesting that the tyrosine catabolic pathway might be the source from which maleylacetate reductase is recruited for the degradation of TFD in pJP4-bearing cells of Pseudomonas sp. strain PKO1.     

Maleylacetate reductase of Pseudomonas sp. strain B13: specificity of substrate conversion and halide elimination.

Maleylacetate reductase (EC 1.3.1.32) plays a major role in the degradation of chloroaromatic compounds by channelling maleylacetate and some chlorinated derivatives into the 3-oxoadipate pathway. Several substituted maleylacetates were prepared in situ by alkaline or enzymatic hydrolysis of dienelactones as the precursor. The conversion of these methyl-, chloro-, fluoro-, and bromo-substituted maleylacetates by malelacetate reductase from 3-chlorobenzoate-grown cells of Pseudomonas sp. strain B13 was studied. Two moles of NADH per mole of substrate was consumed for the conversion of maleylacetates which contain a halogen substituent in the 2 position. In contrast, only 1 mol of NADH was necessary to convert 1 mol of substrates without a halogen substituent in the 2 position. The conversion of 2-fluoro-, 2-chloro-, 2,3-dichloro-, 2,5-dichloro-, 2,3,5-trichloro-, 2-bromo-, 2,3-dibromo-, 2,5-dibromo-, 2-bromo-5-chloro-, 2-chloro-3-methyl-, and 2-chloro-5-methylmaleylacetate was accompanied by the elimination of halide from the 2 position and the temporary occurrence of the corresponding dehalogenated maleylacetate as an intermediate consuming the second mole equivalent of NADH. The properties of the halogen substituents influenced the affinity to the enzyme in the following manner. Km values increased with increasing van der Waals radii and with decreasing electronegativity of the halogen substituents (i.e., low steric hindrance and high electronegativity positively influenced the binding).The Km values obtained with 2-methyl-,3-methyl-, and 5-methylmaleylacetate showed that a methyl substituent negatively affected the affinity in the following order: 2 position >/ = 3 position >> 5 position. A reaction mechanism explaining the exclusive elimination of halogen substituents from the 2 position is proposed.     

Characterization of the Maleylacetate Reductase MacA of Rhodococcus opacus 1CP and Evidence for the Presence of an Isofunctional Enzyme

Maleylacetate reductases (EC 1.3.1.32) have been shown to contribute not only to the bacterial catabolism of some usual aromatic compounds like quinol or resorcinol but also to the degradation of aromatic compounds carrying unusual substituents, such as halogen atoms or nitro groups. Genes coding for maleylacetate reductases so far have been analyzed mainly in chloroaromatic compound-utilizing proteobacteria, in which they were found to belong to specialized gene clusters for the turnover of chlorocatechols or 5-chlorohydroxyquinol. We have now cloned the gene macA, which codes for one of apparently (at least) two maleylacetate reductases in the gram-positive, chlorophenol-degrading strain Rhodococcus opacus 1CP. Sequencing of macA showed the gene product to be relatively distantly related to its proteobacterial counterparts (ca. 42 to 44% identical positions). Nevertheless, like the known enzymes from proteobacteria, the cloned Rhodococcus maleylacetate reductase was able to convert 2-chloromaleylacetate, an intermediate in the degradation of dichloroaromatic compounds, relatively fast and with reductive dehalogenation to maleylacetate. Among the genes ca. 3 kb up- and downstream of macA, none was found to code for an intradiol dioxygenase, a cycloisomerase, or a dienelactone hydrolase. Instead, the only gene which is likely to be cotranscribed with macA encodes a protein of the short-chain dehydrogenase/reductase family. Thus, the R. opacus maleylacetate reductase gene macA clearly is not part of a specialized chlorocatechol gene cluster.Maleylacetate reductases (EC 1.3.1.32) have long been known to be involved in the degradation of chloroaromatic compounds via chlorocatechols as intermediates (10, 31). By reduction of a carbon-carbon double bond they form 3-oxoadipate, a metabolite also of catechol catabolism, and thus compensate for the different oxidation states of chlorinated and nonchlorinated compounds. 2-Chloromaleylacetate, which is formed during turnover of several dichlorocatechols, is initially reductively dechlorinated and then reduced to 3-oxoadipate in a second reaction (22, 47).Corresponding to the biochemical function in chlorocatechol degradation, the following maleylacetate reductase genes have been shown to be associated with dioxygenase, cycloisomerase, and dienelactone hydrolase genes as components of specialized chlorocatechol catabolic operons: tfdF and tfdF_II on pJP4 from the 2,4-dichlorophenoxyacetate-utilizing strain Ralstonia eutropha (Alcaligenes eutrophus) JMP134 (29, 33, 37, 44), tcbF on pP51 from the 1,2,4-trichlorobenzene-degrading strain Pseudomonas sp. strain P51 (45), and clcE from the 3-chlorobenzoate catabolizing strains Pseudomonas sp. strain B13 and Pseudomonas putida AC866(pAC27) (15, 20, 21). Catechol degradation, in contrast, does not require a maleylacetate reductase activity, and corresponding genes do not belong to the known catechol operons. Thus, while at least two of the chlorocatechol catabolic enzymes, i.e., the dioxygenases and cycloisomerases, appear to have been recruited from catechol catabolism, maleylacetate reductase genes must have had a different origin and original function (34).The postulated original function of the maleylacetate reductases is still under discussion. In bacteria, these enzymes have been shown to play a role, for example, in quinol, resorcinol, and 2,4-dihydroxybenzoate degradation (6, 25, 41). Other aromatic growth substrates involving the action of maleylacetate reductase are more exotic, since they carry a fluorine substituent (35), a sulfo group (14), a nitro group (18, 40), or several chlorine substituents (8, 26, 48). Maleylacetate reductase genes have been shown to be part of a specialized gene cluster for 2,4,5-trichlorophenoxyacetate degradation (8, 9) and of a gene cluster for hydroxyquinol conversion which contributes to 4-nitrophenol turnover (4).The chlorocatechol pathway of the chlorophenol-utilizing strain Rhodococcus opacus (erythropolis) 1CP obviously evolved functionally convergent to the corresponding pathway in the proteobacteria mentioned above (13, 39). Thus, it is not surprising that the chlorocatechol gene cluster of strain 1CP is organized differently from the corresponding proteobacterial operons; in fact, its characterization showed that it does not comprise a maleylacetate reductase gene (13). Thus, the nature of the gene cluster(s) encoding a maleylacetate reductase in R. opacus remained to be elucidated. Such gene clusters could complement otherwise incomplete pathways, and they might also have provided the source from which the maleylacetate reductase gene was recruited during evolution of dedicated pathways, such as the proteobacterial chlorocatechol catabolic route.(Some of the results presented here have previously been reported in a preliminary communication [38].)     

Role of tfdC(I)D(I)E(I)F(I) and tfdD(II)C(II)E(II)F(II) gene modules in catabolism of 3-chlorobenzoate by Ralstonia eutropha JMP134(pJP4)

The enzymes chlorocatechol-1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, and maleylacetate reductase allow Ralstonia eutropha JMP134(pJP4) to degrade chlorocatechols formed during growth in 2,4-dichlorophenoxyacetate or 3-chlorobenzoate (3-CB). There are two gene modules located in plasmid pJP4, tfdC(I)D(I)E(I)F(I) (module I) and tfdD(II)C(II)E(II)F(II) (module II), putatively encoding these enzymes. To assess the role of both tfd modules in the degradation of chloroaromatics, each module was cloned into the medium-copy-number plasmid vector pBBR1MCS-2 under the control of the tfdR regulatory gene. These constructs were introduced into R. eutropha JMP222 (a JMP134 derivative lacking pJP4) and Pseudomonas putida KT2442, two strains able to transform 3-CB into chlorocatechols. Specific activities in cell extracts of chlorocatechol-1,2-dioxygenase (tfdC), chloromuconate cycloisomerase (tfdD), and dienelactone hydrolase (tfdE) were 2 to 50 times higher for microorganisms containing module I compared to those containing module II. In contrast, a significantly (50-fold) higher activity of maleylacetate reductase (tfdF) was observed in cell extracts of microorganisms containing module II compared to module I. The R. eutropha JMP222 derivative containing tfdR-tfdC(I)D(I)E(I)F(I) grew four times faster in liquid cultures with 3-CB as a sole carbon and energy source than in cultures containing tfdR-tfdD(II)C(II)E(II)F(II). In the case of P. putida KT2442, only the derivative containing module I was able to grow in liquid cultures of 3-CB. These results indicate that efficient degradation of 3-CB by R. eutropha JMP134(pJP4) requires the two tfd modules such that TfdCDE is likely supplied primarily by module I, while TfdF is likely supplied by module II.