• Journal of Microbiology and Biotechnology
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• v.16 no.12
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• pp.1995-1999
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• 2006

Pseudomonas sp. K82 cultured in p-hydroxybenzoate induces protocatechuate 4,5-dioxygenase (PCD 4,5) for p-hydroxybenzoate degradation. In this study, a 6.0-kbp EcoR1 fragment containing p-hydroxybenzoate degradation genes was cloned from the genome of Pseudomonas sp. K82. Sequence analysis identified four genes, namely, pcaD, pcaA, pcaB, and pcaC genes known to be involved in p-hydroxybenzoate degradation. Two putative 4-hydroxyphenylpyruvate dioxygenases and one putative oxidoreductase were closely located by the p-hydroxybenzoate degradation genes. The gene arrangement and sequences of these p-hydroxybenzoate degradation genes were similar to those of Comamonas testosteroni and Pseudomonas ochraceae. PcaAB (PCD4,5) was overexpressed in the expression vector pGEX-4T-3, purified using a GST column, and confirmed to have protocatechuate 4,5-dioxygenase activity. The N-terminal amino acid sequences of overexpressed PCD4,5 were identical with those of purified PCD4,5 from Pseudomonas sp. K82.

• Journal of Microbiology
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• v.37 no.3
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• pp.123-127
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• 1999

A Pseudomonas sp. strain DJ-12 isolated by 4-cholrobiphenyl enrichment culture technique is capable of utilizing 4-hydroxybenzoic acid as a sole source of carbon and energy. The bacterium catabolized 4-hydroxybenzoic acid through the intermediate formation of protocatechuic acid, which was further metabolized. The cell free extracts of pseudomonas sp. DJ-12, grown on 4-hydroxybenzoic acid showed higher activities of 4-hydroxyenzoate 3-hydroxylase and protocatechuate 4,5-dioxygenase, but the activity of catechnol 2,3-dioxygenase was lower. The results suggest that 4-hydroxybenzoic acid is catabolized via protocatechuic acid rather than catechol or gentisic acid in this bacterium and that the protocatechuic acid formed was metabolized through a metacleavage pathway by protocatechuate 4,5-dioxygenase.

• Journal of Microbiology
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• v.42 no.2
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• pp.152-155
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• 2004

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 dif-ferent 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 puri-fication 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 het-erodimer (${\alpha}$$_1$${\beta}$$_1$). PCD 4,5 exhibits stringent substrate specificity for protocatechuate and its optimal activity occurs at pH 9 and 15 $^{\circ}C$. 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.

• KSBB Journal
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• v.19 no.1
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• pp.50-56
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• 2004

The aim of this work was to investigate the purification and characterization dixoygenases isolated from Delftia sp. JK-2, which could utilize aniline, benzoate, and p-hydroxybenoate as sole carbon and energy source. Catechol 1,2-dioxygenase (C1, 2O), catechol 2,3-dioxygenase(C2, 3O), and protocatechuate 4,5-dioxygenase(4,5-PCD) were isolated by benzoate, aniline, and p-hydroxybenzoate. In initial experiments, several characteristics of C1 ,2O, C2, 3O, and 4,5-PCD separated with ammonium sulfate precipitation, DEAE-sepharose, and Q-sepharose were investigated. Specific activity of C1 ,2O, C2, 3O, and 4,5-PCD were approximately 3.3 unit/mg, 4.7 unit/mg, and 2.0 unit/mg. C1 ,2O and C2, 3O demonstrated their enzyme activities to other substrates, catechol and 4-methylcatechol. 4,5-PCD showed the specific activity to the only substrate, protocatechuate, but the substrates(e.g., catechol, 3-methylcatechol, 4-methylcatechol, 4-chlorocatechol, 4-nitrocatechol) did not show any specific activities in this work. The optimum temperature of C1, 2O, C2, 3O, and 4,5-PCD were 30$^{\circ}C$, and the optimal pHs were approximately 8, 8, and 7, respectively. Ag$\^$+/, Hg$\^$+/, Cu$\^$2+/ showed inhibitory effect on the activity of C1, 2O and C2, 3O, but Ag$\^$+/, Hg$\^$+/, Cu$\^$2+/, Fe$\^$3+/ showed inhibitory effect on the activity of 4,5-PCD. Molecular weight of the C1, 2O, C2, 3O, and 4,5-PCD were determined to approximately 60 kDa,35 kDa, and 62 kDa by SDS-PAGE.

• Korean Journal of Microbiology
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• v.41 no.3
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• pp.195-200
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• 2005

Comamonas sp. strain DJ-12 is a bacterial isolate capable of degrading of 4-chlorobiphenyl (4CB) as a carbon and energy source. The degradation pathway was characterized as being conducted by consecutive reactions of the meta-degradation of 4CB, hydrolytic dechlorination of 4-chlorobenzoate (4CBA), hydroxylation of 4-hydroxybenzoate, and meta-degradation of protocatechuate to product TCA metabolites. The 6.8 kb fragment from the chromosomal DNA of Comamonas sp. strain DJ-12 included the genes encoding for the meta-degradation of PCA; the genes of protocatechuate 4,5-dioxygenase alpha and beta subunits (pmcA and pmcB), 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (pmcC), 2-pyrone-4,6-dicarboxylate hydrolase (pmcD), 4-oxalomesaconate (OMA) hydratase(pmcE), 4-oxalocitramalate (OCM) aldolase (pmcF), and transporter gene (pmcT). They were organized in the order of pmcT-pmcE-pmcF-pmcD-pmcA-pmcB-pmcC. The amino acid sequences deduced from the nucleotide sequences of pmcABCDEFT genes from Comamonas sp. strain DJ-12 exhibited 94 to $98\%$ homologies with those of Comamonas testosteroni BR6020 and Pseudomonas ochraceae NGJ1, but only 52 to $74\%$ with homologies Sphingomonas paucimobilis SYK-6, Sphingomonas sp. LB126, and Arthrobacter keyseri 12B.

• Journal of Life Science
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• v.25 no.5
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• pp.487-495
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• 2015

We carried out pH stability, chemical inhibition, chemical modification, and pH-dependent kinetic parameter assessments to further characterize protocatechuate 3,4-dioxygenase from Pseudomonas pseudoalcaligenes KF707. Protocatechuate 3,4-dioxygenase was stable in the pH range of 4.5~10.5. L-ascorbate and glutathione were competitive inhibitors with $K_{is}$ values of 0.17 mM and 0.86 mM, respectively. DL-dithiothreitol was a noncompetitive inhibitor with a $K_{is}$ value of 1.57 mM and a $K_{ii}$ value of 8.08 mM. Potassium cyanide, p-hydroxybenzoate, and sodium azide showed a noncompetitive inhibition pattern with $K_{is}$ values of 55.7 mM, 0.22 mM, and 15.64 mM, and $K_{ii}$ values of 94.1 mM, 8.08 mM, and 662.64 mM, respectively. $FeCl_{2}$ was the best competitive inhibitor with a $K_{is}$ value of $29{\mu}M$. $FeCl_{3}$, $MnCl_{2}$, $CoCl_{2}$, and $AlCl_{3}$ were also competitive inhibitors with $K_{is}$ values of 1.21 mM, 0.85 mM, 3.98 mM, and 0.21 mM, respectively. Other metal ions showed noncompetitive inhibition patterns. The pH-dependent kinetic parameter data showed that there may be at least two catalytic groups with pK values of 6.2 and 9.4 and two binding groups with pK values of 5.5 and 9.0. Lysine, cysteine, tyrosine, carboxyl, and histidine were modified by their own specific chemical modifiers, indicating that they are involved in substrate binding and catalysis.

• Microbiology and Biotechnology Letters
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• v.23 no.3
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• pp.255-262
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• 1995

Intact cells of Acinetobacter sp. T5-7 completely degraded trichloroethylene (TCE) following growth with phenol. This strain could grow on at least eleven aromatic compounds, e.g., benzaldehyde, benzene, benzoate, benzylalochol, catechol, caffeic acid, 2.4-D, p-hydroxybenzoate, phenol, protocatechuate and salicylate, and did grow on alkane, such as octane. But except phenol, other aromatic compounds did not induced TCE degradation. Phenol biotransformation products, catechol was identified in the culture media. However, catechol-induced cells did not degrade TCE. So we assumed that phenol hydroxylase was responsible for the degradation of TCE. The isolate T5-7 showed growth in MM2 medium containing sodium lactate and catechol rather than phenol, but did not display phenol hydroxyalse activity, suggesting induction of enzyme synthesis by phenol. Phenol hydroxylase activity was independent of added NADH and flavin adenine dinucleotide but was dependent on NADPH addition. Degradation of phenol produced catechols which are then cleaved by meta-fission. We identified catechol-2.3-dioxygenase by active staining of polyacrylamide gel.