Phthalate and 4-hydroxyphthalate metabolism in Pseudomonas testosteroni: purification and properties of 4,5-dihydroxyphthalate decarboxylase.

Phthalate is degraded through 4,5-dihydroxyphthalate and protocatechuate in Pseudomonas testosteroni NH1000. The ezyme 4,5-dihydroxyphthalate decarboxylase, catalyzing the conversion of 4,5-dihydroxyphthalate to protocatechuate and carbon dioxide, was purified approximately 130-fold from phthalate-induced cells of a protocatechuate 4,5-dioxygenase-deficient mutant of P. testosteroni. The most purified preparation showed a single protein band on sodium dodecyl sulfate-acrylamide disc gel electrophoresis with a molecular weight of 38,000. The apparent molecular weight of the native enzyme determined by Sephadex G-200 column chromatography was 150,000. Among the substrate analogs tested, only 4-hydroxyphthalate served as a substrate, which was decarboxylated to form m-hydroxybenzoate. The apparent Km values for 4,5-dihydroxyphthalate and 4-hydroxyphthalate were estimated to be 10.5 micrometer and 1.25 mM, respectively, and the Vmax for the former was 10 times larger than that for the latter. Whereas the wild-type strain could utilize 4-hydroxyphthalate as a sole source of carbon, none of the following could grow with the compound: 4,5-dihydroxyphthalate decarboxylase-deficient, m-hydroxybenzoate-nondegradable, and protocatechuate 4,5-dioxygenase-deficient mutants. Since one-step revertants of these mutants could utilize 4-hydroxyphthalate, the compound appears to be metabolized through m-hydroxybenzoate and protocatechuate in P. testosteroni NH1000.     

Phthalate and 4-hydroxyphthalate metabolism in Pseudomonas testosteroni: purification and properties of 4,5-dihydroxyphthalate decarboxylase.

Phthalate is degraded through 4,5-dihydroxyphthalate and protocatechuate in Pseudomonas testosteroni NH1000. The ezyme 4,5-dihydroxyphthalate decarboxylase, catalyzing the conversion of 4,5-dihydroxyphthalate to protocatechuate and carbon dioxide, was purified approximately 130-fold from phthalate-induced cells of a protocatechuate 4,5-dioxygenase-deficient mutant of P. testosteroni. The most purified preparation showed a single protein band on sodium dodecyl sulfate-acrylamide disc gel electrophoresis with a molecular weight of 38,000. The apparent molecular weight of the native enzyme determined by Sephadex G-200 column chromatography was 150,000. Among the substrate analogs tested, only 4-hydroxyphthalate served as a substrate, which was decarboxylated to form m-hydroxybenzoate. The apparent Km values for 4,5-dihydroxyphthalate and 4-hydroxyphthalate were estimated to be 10.5 micrometer and 1.25 mM, respectively, and the Vmax for the former was 10 times larger than that for the latter. Whereas the wild-type strain could utilize 4-hydroxyphthalate as a sole source of carbon, none of the following could grow with the compound: 4,5-dihydroxyphthalate decarboxylase-deficient, m-hydroxybenzoate-nondegradable, and protocatechuate 4,5-dioxygenase-deficient mutants. Since one-step revertants of these mutants could utilize 4-hydroxyphthalate, the compound appears to be metabolized through m-hydroxybenzoate and protocatechuate in P. testosteroni NH1000.     

Phthalate metabolism in Pseudomonas fluorescens PHK: purification and properties of 4,5-dihydroxyphthalate decarboxylase.

Pseudomonas fluorescens PHK uses 4,5-dihydroxyphthalate as the sole carbon source for o-phthalate catabolism. This intermediate is the substrate for a decarboxylase of the pathway yielding protocatechuate. The decarboxylase was purified to homogeneity by an affinity chromatography procedure in which the reaction product, protocatechuate, was used as a ligand. We describe some properties of the enzyme, including its apparent molecular weight of 420,000 as determined by gel filtration and of 66,000 after sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis, consistent with a hexameric functional protein. The apparent Km for the substrate 4,5-dihydroxyphthalate was 10.4 microM. The characteristics of this enzyme are compared with those described for the isofunctional enzyme from P. testosteroni.     

Utilization of phthalate esters by micrococci

Several strains of Micrococcus have been isolated by enrichment with one of several phthalate esters as sole carbon source. They have been separated into four groups by their esterase content and nutritional characteristics. The catabolic potential for phthalate utilization found in these strains provides further support for designation of the four groups. Pathways for phthalate utilization by 4,5-dihydroxyphthalate and/or 3,4-dihydroxyphthalate and protocatechuate and/or 2,3-dihydroxybenzoate are outlined, which suggests that micrococci possess substantial potential for the catabolism of aromatic compounds.     

Metabolism of dibutylphthalate and phthalate by Micrococcus sp. strain 12B.

Micrococcus sp. strain 12B was isolated by enriching for growth with dibutylphthalate as the sole carbon and energy source. A pathway for the metabolism of dibutylphthalate and phthalate by micrococcus sp. strain 12B is proposed: dibutylphthalate leads to monobutylphthalate leads to phthalate leads to 3,4-dihydro-3,4-dihydroxyphthalate leads to 3,4-dihydroxyphthalate leads to protocatechuate (3,4-dihdroxybenzoate). Protocatechuate is metabolized both by the meta-cleavage pathway through 4-carboxy-2-hydroxymuconic semialdehyde and 4-carboxy-2-hydroxymuconate to pyruvate and oxaloacetate and by the ortho-cleavage pathway to beta-ketoadipate. Dibutylphthalate- and phthalate-grown cells readily oxidized dibutylphthalate, phthalate, 3,4-dihydroxyphthalate, and protocatechuate. Extracts of cells grown with dibutylphthalate or phthalate contained the 3,4-dihydroxyphthalate decarboxylase and the enzymes of the protocatechuater 4,5-meta-cleavage pathway. Extracts of dibutylphthalate-grown cells also contained the protocatechuate ortho-cleavage pathway enzymes. The dibutylphthalate-hydrolyzing esterase and 3,4-dihydroxyphthalate decarboxylase were constitutively synthesized; phthalate-3,4-dioxygenase (and possibly the "dihydrodiol" dehydrogenase) was inducible by phthalate or a metabolite occurring before protocatechuate in the pathway; two protocatechuate oxygenases and subsequent enzymes were inducible by protocatechuate or a subsequent metabolic product. During growth at 37 degrees C, strain 12B gave clones at high frequency that had lost the ability to grow with phthalate esters. One of these nonrevertible mutants, strain 12B-Cl, lacked all of the enzymes required for the metabolism of dibutylphthalate through the protocatechuate meta-cleavage pathway. Enzymes for the metabolism of protocatechuate by the ortho-cleavage pathway were present in this strain grown with p-hydroxybenzoate or protocatechuate.     

An Na+-independent decarboxylation in a marine bacterium: Ion-controlled biotransformations with intact cells

Abstract The catabolism of o -phthalate by a marine Acinetobacter sp. required Na⁺ ions. In contrast, the decarboxylation of 4,5-dihydroxyphthalate, an intermediate in phthalate breakdown, occurred independently of Na⁺ ions and with a quantitative accumulation of the product, protocatechuate. In the presence of Na⁺ ions, protocatechuate did not accumulate and was further oxidized. Transport systems of marine bacteria require Na⁺ ions and extracellular enzymes probably account for Na⁺-independent conversions. Na⁺-independent reactions in marine bacteria present a biotechnological opportunity to use intact cells, rather than cell-free preparations, to effect specific biotransformations.     

Mineralization of 4-sulfophthalate by aPseudomonas strain isolated from the River Elbe

The bacteriumPseudomonas sp. strain RW31 isolated from the river Elbe utilized the ammonium salt of 4-sulfophthalate (4SPA) as sole source of carbon, sulfur, nitrogen, and energy and grew also with phthalate (PA) and several other aromatic compounds as sole carbon and energy source. The xenobiotic sulfo group of 4SPA was eliminated as sulfite, which transiently accumulated in the culture supernatant up to about 10 µM and was slowly oxidized to the stoichiometrical amount of sulfate. Biodegradation routes of 4SPA as well as of PA converged into the protocatechuate pathway and from found activities for the decarboxylation of 4,5-dihydroxyphthalate we deduce this compound the first rearomaticized intermediate after initial dioxygenation. Protocatechuate then underwentmeta-cleavage mediated by a protocatechuate 4,5-dioxygenase activity which was competitively inhibited by the structurally related compound 3,4,5-trihydroxybenzoate; protocatechuate accumulated in the medium up to an about 2 mM concentration. Indications for the presence of selective transport systems are presented.     

Production of 4,5-dihydro-4,5-dihydroxyphthalate from phthalate by a mutant strain of Pseudomonas testosteroni M4-1

Summary Pseudomonas testosteroni M4-1, capable of using phthalate as the sole carbon and energy source, was isolated. Tn5 mutagenesis using pSUP2021 yielded mutant strains of M4-1 that are defective in phthalate metabolism and produce a dihydrodiol compound. The dihydrodiol compound produced by mutant strain M4-122 was isolated and identified as 4,5-dihydro-4,5-dihydroxyphthalate (DDP) by elementary analysis, mass analysis and nuclear magnetic resonance. Various conditions to increase the yield of DDP from phthalate were examined for mutant strain M4-122. With resting cells 6 g DDP/1 were produced. The additional of ethanol to the resting-cell reaction mixture enhanced DDP production and 10 g DDP/1 was produced from 8.3 g/1 of phthalate. Offprint requests to: T. Omori     

Isolation and characterization of marine bacteria capable of utilizing phthalate

Eleven phthalate-degrading bacterial strains were isolated from seawater collected off the coast of Japan. The isolates were found to be most closely related to the marine bacterial genera Alteromonas, Citreicella, Marinomonas, Marinovum, Pelagibaca, Rhodovulum, Sulfitobacter, Thalassobius, Thalassococcus, Thalassospira, and Tropicibacter. For the first time, members of these genera were shown to be capable of growth on phthalate. The plate assay for visual detection of phthalate dioxygenase activity and PCR detection of a possible gene encoding 4,5-dihydroxyphthalate decarboxylase indicated that phthalate is degraded via 4,5-dihydroxyphthalate to protocatechuate in all the isolates.     

Evidence for a novel pathway in the degradation of fluorene by Pseudomonas sp. strain F274.

A fluorene-utilizing microorganism, identified as a species of Pseudomonas, was isolated from soil severely contaminated from creosote use and was shown to accumulate six major metabolites from fluorene in washed-cell incubations. Five of these products were identified as 9-fluorenol, 9-fluorenone, (+)-1,1a-dihydroxy-1-hydro-9-fluorenone, 8-hydroxy-3,4-benzocoumarin, and phthalic acid. This last compound was also identified in growing cultures supported by fluorene. Fluorene assimilation into cell biomass was estimated to be approximately 50%. The structures of accumulated products indicate that a previously undescribed pathway of fluorene catabolism is employed by Pseudomonas sp. strain F274. This pathway involves oxygenation of fluorene at C-9 to give 9-fluorenol, which is then dehydrogenated to the corresponding ketone, 9-fluorenone. Dioxygenase attack on 9-fluorenone adjacent to the carbonyl group gives an angular diol, 1,1a-dihydroxy-1-hydro-9-fluorenone. Identification of 8-hydroxy-3,4-benzocoumarin and phthalic acid suggests that the five-membered ring of the angular diol is opened first and that the resulting 2'-carboxy derivative of 2,3-dihydroxy-biphenyl is catabolized by reactions analogous to those of biphenyl degradation, leading to the formation of phthalic acid. Cell extracts of fluorene-grown cells possessed high levels of an enzyme characteristic of phthalate catabolism, 4,5-dihydroxyphthalate decarboxylase, together with protocatechuate 4,5-dioxygenase. On the basis of these findings, a pathway of fluorene degradation is proposed to account for its conversion to intermediary metabolites. A range of compounds with structures similar to that of fluorene was acted on by fluorene-grown cells to give products consistent with the initial reactions proposed.     

A simple method for detection of enzyme activities involved in the initial step of phthalate degradation in microorganisms

We have developed a simple method for the detection of phthalate 4,5-dioxygenase and 4,5-dihydro-4,5-dihydroxyphthalate dehydrogenase activities in the initial step of phthalate degradation in bacteria. It was found that cells of a Pseudomonas putida strain adapted for phthalate could convert quinolinic acid to a hydroxylated product having λ_max at 315 nm. The occurrence of this compound was visualized by reaction with diazotized p-nitroaniline with which a red compound having λ_max at 512 nm was produced. In practice, if cells in colonies developed on an agar plate containing mineral salt medium supplemented with 0.4% of disodium phthalate and 0.1% of quinolinic acid are active with respect to the 4,5-dihydroxyphthalate pathway, then the colonies would be colored red immediately upon spraying with the diazotized p-nitroaniline reagent. The method was used to identify the phthalate degradative pathway for 27 phthalate-utilizing strains of the genera Pseudomonas (18 strains), Agrobacterium (3 strains), Alcaligenes (5 strains), and Micrococcus (1 strain). It was found that 24 of the 26 Gram-negative bacteria have the 4,5-dihydroxyphthalate pathway and that the remaining two strains of Pseudomonas sp. may metabolize via an unidentified pathway other than the dihydroxyphthalate pathways, and the Gram-positive strain of Micrococcus sp. metabolizes phthalate via the 3,4-dihydroxyphthalate pathway.     

Production of 3,4-dihydroxyphthalate from phthalate by a membrane-bound two-enzyme system from Rhodococcus erythropolis

 Gram-positive Rhodococcus erythropolis strain S1 formed enzymes for the degradation of phthalate when grown in a phthalate-containing minimal medium. The membrane fraction prepared from phthalate-grown cells by ultrasonication converted phthalate to protocatechuate as the final product. Using two membrane-bound enzymes, phthalate 3,4-dioxygenase (PO) and 3,4-dihydro-3,4-dihydroxyphthalate 3,4-dehydrogenase (PH), prepared by solubilization of the membrane fraction, 3,4-dihydroxyphthalate was selectively obtained from phthalata. Fe²⁺ and Mn²⁺ stimulated the formation of 3,4-dihydroxyphthalate by the membrane-bound PO and PH system. Received: 27 April 1994/Received last revision: 19 August 1994/Accepted: 12 September 1994     

Plasmid-encoded phthalate catabolic pathway in Arthrobacter keyseri 12B

Several 2-substituted benzoates (including 2-trifluoromethyl-, 2-chloro-, 2-bromo-, 2-iodo-, 2-nitro-, 2-methoxy-, and 2-acetyl-benzoates) were converted by phthalate-grown Arthrobacter keyseri (formerly Micrococcus sp.) 12B to the corresponding 2-substituted 3,4-dihydroxybenzoates (protocatechuates). Because these products lack a carboxyl group at the 2 position, they were not substrates for the next enzyme of the phthalate catabolic pathway, 3,4-dihydroxyphthalate 2-decarboxylase, and accumulated. When these incubations were carried out in iron-containing minimal medium, the products formed colored chelates. This chromogenic response was subsequently used to identify recombinant Escherichia coli strains carrying genes encoding the responsible enzymes, phthalate 3,4-dioxygenase and 3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase, from the 130-kbp plasmid pRE1 of strain 12B. Beginning with the initially cloned 8.14-kbp PstI fragment of pRE824 as a probe to identify recombinant plasmids carrying overlapping fragments, a DNA segment of 33.5 kbp was cloned from pRE1 on several plasmids and mapped using restriction endonucleases. From these plasmids, the sequence of 26,274 contiguous bp was determined. Sequenced DNA included several genetic units: tnpR, pcm operon, ptr genes, pehA, norA fragment, and pht operon, encoding a transposon resolvase, catabolism of protocatechuate (3,4-dihydroxybenzoate), a putative ATP-binding cassette transporter, a possible phthalate ester hydrolase, a fragment of a norfloxacin resistance-like transporter, and the conversion of phthalate to protocatechuate, respectively. Activities of the eight enzymes involved in the catabolism of phthalate through protocatechuate to pyruvate and oxaloacetate were demonstrated in cells or cell extracts of recombinant E. coli strains.     

Enzyme complex structure and location of enzymes in the phthalate degradative pathway in Rhodococcus erythropolis

A. SUEMORI, K. NAKAJIMA, R. KURANE AND Y. NAKAMURA. 1996. Rhodococcus erythropolis strain S1 formed enzymes essential to the degradation of phthalate when grown in phthalate-minimal medium. The reaction responsible for the dihydroxylation of the phthalate-benzene ring was concluded to be catalysed by membrane-associated phthalate 3,4-dioxygenase (PO). Of the other enzymes involved, 3,4-dihydro-3,4-dihydroxyphthalate 3,4-dehydrogenase (PH) and 3,4-dihydroxyphthalate 2-decarboxylase (PC) appeared likely to be membrane-bound, while protocatechuate 3,4-dioxygenase appeared to be present in the cytoplasm. Based on the data, the membrane-bound PO and PH apparently form an enzyme complex, which is associated with the NADH-regenerating system.     

Bacterial Decarboxylation of o-Phthalic Acids

The decarboxylation of phthalic acids was studied with Bacillus sp. strain FO, a marine mixed culture ON-7, and Pseudomonas testosteroni. The mixed culture ON-7, when grown anaerobically on phthalate but incubated aerobically with chloramphenicol, quantitatively converted phthalic acid to benzoic acid. Substituted phthalic acids were also decarboxylated: 4,5-dihydroxyphthalic acid to protocatechuic acid; 4-hydroxyphthalic and 4-chlorophthalic acids to 3-hydroxybenzoic and 3-chlorobenzoic acids, respectively; and 3-fluorophthalic acid to 2-and 3-fluorobenzoic acids. Bacillus sp. strain FO gave similar results except that 4,5-dihydroxyphthalic acid was not metabolized, and both 3- and 4-hydroxybenzoic acids were produced from 4-hydroxyphthalic acid. P. testosteroni decarboxylated 4-hydroxyphthalate (to 3-hydroxybenzoate) and 4,5-dihydroxyphthalate but not phthalic acid and halogenated phthalates. Thus, P. testosteroni and the mixed culture ON-7 possessed 4,5-dihydroxyphthalic acid decarboxylase, previously described in P. testosteroni, that metabolized 4,5-dihydroxyphthalic acid and specifically decarboxylated 4-hydroxyphthalic acid to 3-hydroxybenzoic acid. The mixed culture ON-7 and Bacillus sp. strain FO also possessed a novel decarboxylase that metabolized phthalic acid and halogenated phthalates, but not 4,5-dihydroxyphthalate, and randomly decarboxylated 4-hydroxyphthalic acid. The decarboxylation of phthalic acid is suggested to involve an initial reduction to 1,2-dihydrophthalic acid followed by oxidative decarboxylation to benzoic acid.     

2-pyrone-4,6-dicarboxylic acid, a catabolite of gallic acids in Pseudomonas species.

2-Pyrone-4,6-dicarboxylate hydrolase was purified from 4-hydroxybenzoate-grown Pseudomonas testosteroni. Gel filtration and electrophoretic measurements indicated that the preparation was homogeneous and gave a molecular weight of 37,200 for the single subunit of the enzyme. Hydrolytic activity was dependent upon a functioning sulfhydryl group(s) and was freely reversible; the equilibrium position was dependent upon pH, with equimolar amounts of pyrone and open-chain form present at pH 7.9. Since the hydrolase was strongly induced when the nonfluorescent organisms P. testosteroni and P. acidovorans grew with 4-hydroxybenzoate, it is suggested that 2-pyrone-4,6-dicarboxylate is a normal intermediate in the meta fission degradative pathway of protocatechuate. Laboratory strains of fluorescent pseudomonads did not metabolize 2-pyrone-4,6-dicarboxylate, but a strain of P. putida was isolated from soil that utilized this compound for growth; the hydrolase was then induced, but it was absent from extracts of 4-hydroxybenzoate-grown cells that readily catabolized protocatechuate by ortho fission reactions. 2-Pyrone-4,6-dicarboxylic acid was the major product formed when gallic acid was oxidized by purified protocatechuate 3,4-dioxygenase. Protocatechuate 4,5-dioxygenase gave only the open-chain ring fission product when gallic acid was oxidized, but the enzyme attacked 3-O-methylgallic acid, giving 2-pyrone-4,6-dicarboxylic acid as the major product. Cell suspensions of 4-hydroxybenzoate-grown P. testosteroni readily oxidized 3-O-methylgallate with accumulation of methanol.     

Absolute Configuration of a Metabolite in the m-Fission Pathway of Protocatechuate

An aldolase, which is induced in Pseudomonas testosteroni during growth with p-hydroxybenzoate, preferentially attacks the R form of 4-hydroxy-4-methyl-2-oxoglutarate, a metabolite of protocatechuate catabolism.     

Phthalate catabolic gene cluster is linked to the angular dioxygenase gene in <Emphasis Type="Italic">Terrabacter</Emphasis> sp. strain DBF63

Phthalate is a metabolic intermediate of the pathway of fluorene (FN) degradation via angular dioxygenation. A gene cluster responsible for the conversion of phthalate to protocatechuate was cloned from the dibenzofuran (DF)- and FN-degrading bacterium Terrabacter sp. strain DBF63 and sequenced. The genes encoding seven catabolic enzymes, oxygenase large subunit of phthalate 3,4-dioxygenase (phtA1), oxygenase small subunit of phthalate 3,4-dioxygenase (phtA2), cis-3,4-dihydroxy-3,4-dihydrophthalate dehydrogenase (phtB), [3Fe-4S] or [4Fe-4S] type of ferredoxin (phtA3), ferredoxin reductase (phtA4), 3,4-dihydroxyphthalate decarboxylase (phtC) and putative regulatory protein (phtR), were found in the upstream region of the angular dioxygenase gene (dbfA1A2), encoded in this order. Escherichia coli carrying phtA1A2BA3A4 genes converted phthalate to 3,4-dihydroxyphthalate, and the 3,4-dihydroxyphthalate decarboxylase activity by E. coli cells carrying phtC was finally detected with the introduction of a Shine-Dalgarno sequence in the upstream region of its initiation codon. Homology analysis on the upstream region of the pht gene cluster revealed that there was an insertion sequence (IS) (ISTesp2; ORF14 and its flanking region), part of which was almost 100% identical to the orf1 and its flanking region adjacent to the extradiol dioxygenase gene ( bphC1) involved in the DF degradation of Terrabacter sp. strain DPO360 [Schmid et al. (1997) J Bacteriol 179:53-62]. This suggests that ISTesp2 plays a role in the metabolism of aromatic compounds in Terrabacter sp. strains DBF63 and DPO360.     

Molecular characterization of the genes pcaG and pcaH, encoding protocatechuate 3,4-dioxygenase, which are essential for vanillin catabolism in Pseudomonas sp. strain HR199

Pseudomonas sp. strain HR199 is able to utilize eugenol (4-allyl-2-methoxyphenol), vanillin (4-hydroxy-3-methoxybenzaldehyde), or protocatechuate as the sole carbon source for growth. Mutants of this strain which were impaired in the catabolism of vanillin but retained the ability to utilize eugenol or protocatechuate were obtained after nitrosoguanidine mutagenesis. One mutant (SK6169) was used as recipient of a Pseudomonas sp. strain HR199 genomic library in cosmid pVK100, and phenotypic complementation was achieved with a 5.8-kbp EcoRI fragment (E58). The amino acid sequences deduced from two corresponding open reading frames (ORF) identified on E58 revealed high degrees of homology to pcaG and pcaH, encoding the two subunits of protocatechuate 3,4-dioxygenase. Three additional ORF most probably encoded a 4-hydroxybenzoate 3-hydroxylase (PobA) and two putative regulatory proteins, which exhibited homology to PcaQ of Agrobacterium tumefaciens and PobR of Pseudomonas aeruginosa, respectively. Since mutant SK6169 was also complemented by a subfragment of E58 that harbored only pcaH, this mutant was most probably lacking a functional beta subunit of the protocatechuate 3, 4-dioxygenase. Since this mutant was still able to grow on protocatechuate and lacked protocatechuate 4,5-dioxygenase and protocatechuate 2,3-dioxygenase, the degradation had to be catalyzed by different enzymes. Two other mutants (SK6184 and SK6190), which were also impaired in the catabolism of vanillin, were not complemented by fragment E58. Since these mutants accumulated 3-carboxy muconolactone during cultivation on eugenol, they most probably exhibited a defect in a step of the catabolic pathway following the ortho cleavage. Moreover, in these mutants cyclization of 3-carboxymuconic acid seems to occur by a syn absolute stereochemical course, which is normally only observed for cis, cis-muconate lactonization in pseudomonads. In conclusion, vanillin is degraded through the ortho-cleavage pathway in Pseudomonas sp. strain HR199 whereas protocatechuate could also be metabolized via a different pathway in the mutants.     

Characterization of protocatechuate 4,5-dioxygenase induced from p-hydroxybenzoate-cultured Pseudomonas sp. K82

Pseudomonas sp. K82 has been reported to be an aniline-assimilating soil bacterium. However, this strain can use not only aniline as a sole carbon and energy source, but can also utilize benzoate, p-hydroxybenzoate, and aniline analogues. The strain accomplishes this metabolic diversity by using different aerobic pathways. Pseudomonas sp. K82, when cultured in p-hydroxybenzoate, showed extradiol cleavage activity of protocatechuate. In accordance with those findings, our study attempted the purification of protocatechuate 4,5-dioxygenase (PCD 4,5). However the purified PCD 4,5 was found to be very unstable during purification. After Q-sepharose chromatography was performed, the crude enzyme activity was augmented by a factor of approximately 4.7. From the Q-sepharose fraction which exhibited PCD 4,5 activity, two subunits of PCD4,5 (alpha subunit and beta subunit) were identified using the N-terminal amino acid sequences of 15 amino acid residues. These subunits were found to have more than 90% sequence homology with PmdA and PmdB of Comamonas testosteroni. The molecular weight of the native enzyme was estimated to be approximately 54 kDa, suggesting that PCD4,5 exists as a heterodimer (alpha1beta1). PCD 4,5 exhibits stringent substrate specificity for protocatechuate and its optimal activity occurs at pH 9 and 15 degrees C. PCR amplification of these two subunits of PCD4,5 revealed that the alpha subunit and beta subunit occurred in tandem. Our results suggest that Pseudomonas sp. K82 induced PCD 4,5 for the purpose of p-hydroxybenzoate degradation.