• Toxicological Research
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• v.24 no.2
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• pp.101-107
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• 2008

Studies on hypoxia-signaling pathways have revealed novel Fe(II) and $\alpha$-ketoglutarate-dependent dioxygenases that hydroxylate prolyl or asparaginyl residues of a transactivator, Hypoxia-Inducible $Factor-\alpha(HIF-\alpha)$ protein. The recognition of these unprecedented dioxygenases has led to open a new paradigm that the hydroxylation mediates an instant post-translational modification of a protein in response to the changes in cellular concentrations of oxygen, reducing agents, or $\alpha$-ketoglutarate. Activity of $HIF-\alpha$ is repressed by two hydroxylases. One is $HIF-\alpha$ specific prolyl-hydroxylases, referred as prolyl-hydroxylase domain(PHD). The other is $HIF-\alpha$ specific asparaginyl-hydroxylase, referred as factor-inhibiting HIF-1(FIH-1). The facts (i) that many dioxygenases commonly use molecular oxygen and reducing agents during detoxification of xenobiotics, (ii) that detoxification reaction produces radicals and reactive oxygen species, and (iii) that activities of both PHD and FIH-1 are regulated by the changes in the balance between oxygen species and reducing agents, imply the possibility that the activity of $HIF-\alpha$ can be increased during detoxification process. The importance of $HIF-\alpha$ in cancer and ischemic diseases has been emphasized since its target genes mediate various hypoxic responses including angiogenesis, erythropoiesis, glycolysis, pH balance, metastasis, invasion and cell survival. Therefore, activators of PHDs and FIH-1 can be potential anticancer drugs which could reduce the activity of HIF, whereas inhibitors, for preventing ischemic diseases. This review highlights these novel dioxygenases, PHDs and FIH-1 as specific target against not only cancers but also ischemic diseases.

• Bulletin of the Korean Chemical Society
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• v.21 no.9
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• pp.923-928
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• 2000

[FeIII(BBA)DBC]ClO4 as a new functional model for catechol dioxygenases has been synthesized, where BBA is a bis(benzimidazolyl-2-methyl)amine and DBC is a 3,5-di-tert-butylcatecholate dianion.The BBA complex has a structuralfeature that iron cent er has a five-coordinate geometry similar to that of catechol dioxygenase-substrate complex.The BBA complex exhibits strong absorptionbands at 560 and 820 nm in CH3CN which are assigned to catecholate to Fe(III) charge transfer transitions. It also exhibits EPR signals at g = 9.3 and 4.3 which are typical values for the high-spin FeIII (S = 5/2) complex with rhombicsymmetry. Interestingly, the BBA complex reacts with O2 within an hour to afford intradiol cleavage (35%) and extradiol cleavage (60%) products. Surprisingly, a green color intermediate is observed during the oxygenation process of the BBA com-plex in CH3CN. This green intermediate shows a broad isotropic EPR signal at g = 2.0. Based on the variable temperature EPR study, this isotropic signalmight be originated from the [Fe(III)-peroxo-catecholate] species havinglow-spin FeIII center, not from the simple organic radical. Consequently,it allows O2 to bind to iron cen-ter forming the Fe(III)-superoxide species that converts to the Fe(III)-peroxide intermediate. These present data can lead us tosuggest that the oxygen activation mechanism take place for the oxidative cleavingcatechols of the five-coordinate model systems for catechol dioxygenases.

• Bulletin of the Korean Chemical Society
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• v.20 no.12
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• pp.1428-1432
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• 1999

[Fe$^{II}$(BLPA)DBCH]BPh₄ (1), a new functional model for the extradiol-cleaving catechol dioxygenases, has been synthesized, where BLPA is bis(6-methyl-2-pyridylmethyl)(2-pyridylmethyl)amine and DBCH is 3,5-di-tert-butylcatecholate monoanion. ¹H NMR and EPR studies confirm that 1 has a high-spin Fe(II) (S = 2) center. The electronic spectrum of 1 exhibits one absorption band at 386 nm, showing the yellow color of the typical [Fe$^{II}$(BLPA)] complex. Upon exposure to O₂, 1 is converted to an intense blue species within a minute. This blue species exhibits two intense bands at 586 and 960 nm and EPR signals at g = 5.5 and 8.0 corresponding to the high-spin Fe(III) complex (S = 5/2, E/D = 0.11). This blue complex further reacts with O₂ to be converted to (μ-oxo)Fe$^{III}_2$ complex within a few hours. Interestingly, 1 affords intradiol cleavage (65%) and extradiol cleavage (20%) products after the oxygenation. It can be suggested that 1 undergoes two different oxygenation pathways. The one takes the substrate activation mechanism proposed for the intradiol cleavage products after the oxidation of the $Fe^II\;to\;Fe^{III}$. The other involves the direct attack of O₂ to $Fe^{II}$ center, forming the $Fe^{III}$-superoxo intermediate which can give rise to the extradiol cleavage products. 1 is the first functional Fe(II) complex for extradiol-cleaving dioxygenases giving extradiol cleavage products.

• Bulletin of the Korean Chemical Society
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• v.18 no.11
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• pp.1166-1172
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• 1997

[FeⅢ(BLPA)DBC]BPh4, a new functional model for the catechol dioxygenases, has been synthesized, where BLPA is bis((6-methyl-2-pyridyl)methyl)(2-pyridylmethyl)amine and DBC is 3,5-di-tert-butylcatecholate dianion. The BLPA complex has a structural feature that iron center has a six-coordinate geometry with N4O2 donor set. It exhibits EPR signals at g=5.5 and 8.0 which are typical values for the high-spin FeⅢ (S=5/2) complex with axial symmetry. The BLPA complex reacts with O2 within a few hours to afford intradiol cleavage (75%) and extradiol cleavage (15%) products which is very unique result of all [Fe(L)DBC] complexes studied. The iron-catecholate interaction of BLPA complex is significantly stronger, resulting in the enhanced covalency of the metal-catecholate bonds and low energy catecholate to FeⅢ charge transfer bands at 583 and 962 nm in CH3CN. The enhanced covalency is also reflected by the isotropic shifts exhibited by the DBC protons, which indicate increased semiquinone character. The greater semiquinone character in the BLPA complex correlates well with its high reactivity towards O2. Kinetic studies of the reaction of the BLPA complex with 1 atm O2 in CH3OH and CH2Cl2 under pseudo-first order conditions show that the BLPA complex reacts with O2 much slower than the TPA complex, where TPA is tris(2-pyridylmethyl)amine. It is presumably due to the steric effect of the methyl substituent on the pyridine ring. Nevertheless, both the high specificity and the fast kinetics can be rationalized on the basis of its low energy catecholate to FeⅢ charge transfer bands and large isotropic NMR shifts for the BLPA protons. These results provide insight into the nature of the oxygenation mechanism of the catechol dioxygenases.

• Proceedings of the Zoological Society Korea Conference
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• 1997.10b
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• pp.274.2-274
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• 1997

No Abstract, See Full Text

• BMB Reports
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• v.32 no.4
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• pp.399-404
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• 1999

2,3-Dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD), which catalyzes the ring meta-cleavage of 2,3-dihydroxybiphenyl, is encoded by the phnQ gene of biphenyl- and phenanthrene-degrading Pseudomonas sp. strain DJ77. We determined the nucleotide sequence of a DNA fragment of 1497 base pairs which included the phnQ gene. The fragment lncluded an open reading frame of 903 base pairs to accommodate the enzyme. The predicted amino acid sequence of the enzyme subunit consisted of 300 residues. In front of the gene, a sequence resembling an E. coli promoter was identified, which led to constitutive expression of the cloned gene in E. coli. The deduced amino acid sequence of the PhnQ enzyme exhibited 85.6% identity with that of the corresponding enzyme in Sphingomonas yanoikuyae Q1 (formerly S. paucimobilis Q1) and 22.1% identity with that of catechol 1,2,3-dioxygenase from the same DJ77 strain. PhnQ showed broader substrate preference than previously-cloned PhnE, catechol 2,3-dioxygenase. Ten amino acid residues, considered to be important for the role of extradiol dioxygenases, were conserved.

• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.249-254
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• 1999

A catechol 2,3-dioxygenase (C23O) was purified to apparent homogeneity from Pseudomonas putida SU10 through several purification steps consisting of ammonium sulfate precipitation and chromatographies on DEAE 5PW, Superdex S-200, and Resource-Q. Gel filtration indicated a molecular mass under nondenaturing conditions of about 130 kDa. The enzyme has a subunit of 34 kDa as was determined by SDS-PAGE. These results suggest that the native enzyme is composed of four identical subunits. The N-terminal amino acid sequence (30 residues) of the enzyme has been determined and exhibits high identity with other extradiol dioxygenases. The reactivity of this enzyme towards catechol and methyl-substituted catechols is somewhat different from that seen for other catechol 2,3-dioxygenases, with 4-methylcatechol cleaved at a higher rate than catechol or 3-methylcatechol. $K_m$ values of the enzyme for these substrates are between 3.5 and 5.7 M.

• Journal of Microbiology
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• v.38 no.1
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• pp.40-43
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• 2000

The aromatic dioxygenase system in Sphingomonas yanoikuyae strain Bl consists of three components, an oxygenase, a ferredoxin, and a reductase. The insertional knockout of the bphA4 gene encoding a reductase and subsequent complementation experiments showed that the reductase encoded by bphA4 in S. yanoikuyae strain Bl is associated with multiple dioxygenase components including that of toluate dioxygenase (XyIXY).

• Journal of Microbiology and Biotechnology
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• v.16 no.5
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• pp.778-785
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• 2006

Catechol 1,2-dioxygenase was purified from cells of R. rhodochrous N75 grown at the expense of benzoate and p-toluate as the sole sources of carbon. A single catechol 1,2-dioxygenase was found to be induced with either growth substrate. The enzyme has an estimated $M_r$ of 71,000 consisting of two identical subunits. Catechol 1,2-dioxygenase from R. rhodochrous N75 exhibits some unusual properties including: broad substrate specificity, extradiol cleavage activity with 4-methylcatechol and low $K_m$ values for halocatechols, suggesting that this enzyme is distinct from other known catechol and chlorocatechol 1,2-dioxygenases.

• Journal of Microbiology and Biotechnology
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• v.12 no.1
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• pp.172-175
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• 2002

The complete nucleotide sequence of the nahC gene from Pseudomonas fluorescens, the structural gene for 1,2-dihydroxynaphthalene (1,2-DHN) dioxygenase, was determined. The 1,2-DHN dioxygenase is an extradiol ring-cleavage enzyme that cleaves the first ring of 1,2-dihydroxynaphthalene. The amino acid sequence of the dioxygenase deduced from the nucleotide sequence suggested that the holoenzyme consists of eight identical subunits with a molecular weight of approximately 34,200. The amino acid sequence of 1,2-DHN dioxygenase showed more than $90\%$ homology with those of the dioxygenases of other Pseudomonas strains. However, sequence similarity with those of the Sphingomonas species was less than $60\%$. The nahC gene of P. fluorescens was moderately expressed in E. coli NM522, as determined by enzymatic activity.