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At least 19 recordsLinked to original sources

PubMed1996

Oxidation of 6,7-dihydro-5H-benzocycloheptene by bacterial strains expressing naphthalene dioxygenase, biphenyl dioxygenase, and toluene dioxygenase yields homochiral monol or cis-diol enantiomers as major products.

Bacterial strains expressing naphthalene, biphenyl, and toluene dioxygenase were examined for their abilities to oxidize 6,7-dihydro-5H-benzocycloheptene (benzocyclohept-1-ene). The major oxidation products were isolated, and their absolute configurations were determined by chiral 1H nuclear magnetic resonance analysis of diastereomeric boronate esters, chiral stationary-phase high-pressure liquid chromatography, and stereo-chemical correlation. Pseudomonas sp. strain 9816/11 and Sphingomonas yanoikuyae (formerly identified as a Beijerinckia sp.) B8/36 expressing naphthalene and biphenyl dioxygenases, respectively, oxidized benzocyclohept-1-ene to a major product identified as (-)-(1R,2S)-cis-dihydroxybenzocycloheptane (> 98% enantiomeric excess [ee], 50 and 90% yield, respectively). In contrast, Pseudomonas putida F39/D expressing toluene dioxygenase oxidized benzocyclohept-1-ene to (+)-(5R)-hydroxybenzocyclohept-1-ene (> 98% ee, 90% yield) as the major metabolite and to the "opposite" diol, (+)-(1S,2R)-cis-dihydroxybenzocycloheptane (> 98% ee, 10% yield). The results indicate that, for benzocyclohept-1-ene, the major reaction catalyzed by naphthalene and biphenyl dioxygenases is dioxygenation whereas toluene dioxygenase catalyzes mainly R-stereospecific benzylic monooxygenation. Although the type of reaction catalyzed by each organism was not predictable, the absolute configuration of the diol and monol products formed by naphthalene and toluene dioxygenases are consistent with the stereochemistry of the products formed by these enzymes from other benzocycloalkene substrates.

Bacteria↗

PubMed1985

[17O]Water and nitric oxide binding by protocatechuate 4,5-dioxygenase and catechol 2,3-dioxygenase. Evidence for binding of exogenous ligands to the active site Fe2+ of extradiol dioxygenases.

Pseudomonas testosteroni protocatechuate 4,5-dioxygenase and Pseudomonas putida catechol 2,3-dioxygenase (metapyrocatechase) catalyze extradiol-type oxygenolytic cleavage of the aromatic ring of their substrates. The essential active site Fe2+ of each enzyme binds nitric oxide (NO) to produce an EPR active complex with an electronic spin of S = 3/2. Hyperfine broadening of the EPR resonances of the nitrosyl complexes by 17O-enriched H2O shows that water is bound directly to the Fe2+ in the native enzymes, but is apparently displaced in substrate complexes. NO is not displaced by either substrates or inhibitors. The EPR spectra of several enzyme-inhibitor-NO complexes are different from those of enzyme-NO or enzyme-substrate-NO complexes and are found to be broadened by 17O-enriched water. The data show that at least 2 and perhaps 3 sites in the Fe ligation can be occupied by exogenous ligands. Furthermore, it is likely that substrates and inhibitors displace water by binding either at or near to the Fe in the nitrosyl complex. Nitric oxide binding is found to be substrate-dependent for each enzyme. Native catechol 2,3-dioxygenase exhibits KD values of 190 microM and 2.0 mM for NO binding in two types of independent sites. Only one type of site is observed in the catechol complex which exhibits a KD for NO of 3.4 microM. One type of NO binding site is observed for both the native and substrate complexed protocatechuate 4,5-dioxygenase with KD values of 360 and 3 microM, respectively. The presence of a specific site in the Fe coordination for NO which is modified in the substrate complex, suggests that O2 binding by the extradiol dioxygenases may also occur at the Fe.

Binding Sites↗

PubMed1996

2,4-dioxygenases catalyzing N-heterocyclic-ring cleavage and formation of carbon monoxide. Purification and some properties of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase from Arthrobacter sp. Rü61a and comparison with 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase from Pseudomonas putida 33/1.

1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (MeQDO) was purified from quinaldine-grown Arthrobacter sp. Rü61a. It was enriched 59-fold in a yield of 22%, and its properties were compared with 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) purified from Pseudomonas putida 33/1. The enzyme-catalyzed conversions were performed in an (18O)O2/(16O)O2 atmosphere. Two oxygen atoms of either (18O)O2 or (16O)O2 were incorporated at C2 and C4 of the respective substrates, indicating that these unusual enzymes, which catalyze the cleavage of two carbon-carbon bonds concomitant with CO formation, indeed are 2,4-dioxygenases. Both enzymes are small monomeric proteins of 32 kDa (MeQDO) and 30 kDa (QDO). The apparent K(m) values of MeQDO for 1H-3-hydroxy-4-oxoquinaldine and QDO for 1H-3-hydroxy-4-oxoquinoline were 30 microM and 24 microM, respectively. In both 2,4-dioxygenases, there was no spectral evidence for the presence of a chromophoric cofactor. EPR analyses of MeQDO did not reveal any signal that could be assigned to an organic radical species or to a metal, and X-ray fluorescence spectrometry of both enzymes did not show any metal present in stoichiometric amounts. Ethylxanthate, metal-chelating agents (tiron, alpha, alpha'-bipyridyl, 8-hydroxyquinoline, o-phenanthroline, EDTA, diphenylthiocarbazone, diethyldithiocarbamate), reagents that modify sulfhydryl groups (iodoacetamide, N-ethylmaleimide, p-hydroxymercuribenzoate), and reducing agents (sodium dithionite, dithiothreitol, mercaptoethanol) either did not affect 2,4-dioxygenolytic activities at all or inhibited at high concentrations only. With respect to the supposed lack of any cofactor and with respect to the inhibitors of dioxygenolytic activities, MeQDO and QDO resemble aci-reductone oxidase (CO-forming) from Klebsiella pneumoniae, which catalyzes 1,3-dioxygenolytic cleavage of 1,2-dihydroxy-3-keto-S-methylthiopentene anion (Wray, J. W. & Abeles, R. H. (1993) J. Biol. Chem. 268, 21466-21469; Wray, J. W. & Abeles, R. H. (1995) J. Biol. Chem. 270, 3147-3153). 1H-3-Hydroxy-4-oxoquinaldine and 1H-3-hydroxy-4-oxoquinoline were reactive towards molecular oxygen in the presence of the base catalyst potassium-tert.-butoxide in the aprotic solvent N,N-dimethylformamide. Base-catalyzed oxidation, yielding the same products as the enzyme-catalyzed conversions, provides a non-enzymic model reaction for 2,4-dioxygenolytic release of CO from 1H-3-hydroxy-4-oxoquinaldine and 1H-3-hydroxy-4-oxoquinoline.

Arthrobacter↗

PubMed1999

Genetic and biochemical comparison of 2-aminophenol 1,6-dioxygenase of Pseudomonas pseudoalcaligenes JS45 to meta-cleavage dioxygenases: divergent evolution of 2-aminophenol meta-cleavage pathway.

Nitrobenzene is degraded to pyruvate and acetaldehyde by Pseudomonas pseudoalcaligenes JS45 via a reductive pathway, and by Comamonas sp. JS765 via an oxidative pathway. Although the initial reactions in the degradation of nitrobenzene by the two bacteria are totally different, the lower pathways are similar and converge at the level of 4-oxalocrotonate. In order to further investigate the biochemical properties and reveal the evolutionary relationships between the two lower pathways, the genes encoding the 2-aminophenol 1,6-dioxygenase were cloned and sequenced. 2-Aminophenol 1,6-dioxygenase from P. pseudoalcaligenes JS45 and catechol 2,3-dioxygenase from Comamonas sp. JS765 were able to act on both catechol and 2-aminophenol, but catechol was a suicide substrate of 2-aminophenol 1,6-dioxygenase. The activity of 2-aminophenol 1,6-dioxygenase was restored after removal of catechol and incubation with ascorbate and FeCl(2). Both the alpha-subunit (AmnA) and the beta-subunit (AmnB) of the dioxygenase from P. pseudoalcaligenes JS45 show a high degree of identity to the corresponding subunits of the ring-fission dioxygenase from Pseudomonas sp. AP-3: 67% for the alpha-subunit, and 84% for the beta-subunit. Sequence similarity studies suggest that the beta-subunits of both 2-aminophenol 1,6-dioxygenases are distantly related to homoprotocatechuate 2,3-dioxygenase from Escherichia coli strains W and C and then to catechol 2, 3-dioxygenase from Alcaligenes eutrophus. Four active-site-relevant histidines are conserved in AmnB, but not in AmnA. The lack of conserved histidines indicates the absence of an Fe(2+) binding site in AmnA, which explains the previous observations of only approximately one Fe(2+) per two subunits in the 2-aminophenol 1, 6-dioxygenases from P. pseudoalcaligenes JS45. The 2-aminophenol 1, 6-dioxygenase genes are located upstream of the 2-aminomuconic semialdehyde dehydrogenase gene, and a putative member of the YjgF protein family is upstream of the dioxygenase genes. Transcriptional analysis indicates that the YjgF-like protein, 2-aminophenol 1, 6-dioxygenase, and 2-aminomuconic semialdehyde dehydrogenase are coordinately transcribed. A putative ORF similar to part of the RNA helicase genes is downstream of the dehydrogenase gene. Both the novel organization of the genes and the phylogeny of the dioxygenases and dehydrogenase indicate that the 2-aminophenol pathway in P. pseudoalcaligenes JS45 represents an example of a distant divergent evolution of meta-cleavage pathways.

Amino Acid Sequence↗

PubMed1995

A manganese-dependent dioxygenase from Arthrobacter globiformis CM-2 belongs to the major extradiol dioxygenase family.

Almost all bacterial ring cleavage dioxygenases contain iron as the catalytic metal center. We report here the first available sequence for a manganese-dependent 3,4-dihydroxyphenylacetate (3,4-DHPA) 2,3-dioxygenase and its further characterization. This manganese-dependent extradiol dioxygenase from Arthrobacter globiformis CM-2, unlike iron-dependent extradiol dioxygenases, is not inactivated by hydrogen peroxide. Also, ferrous ions, which activate iron extradiol dioxygenases, inhibit 3,4-DHPA 2,3-dioxygenase. The gene encoding 3,4-DHPA 2,3-dioxygenase, mndD, was identified from an A. globiformis CM-2 cosmid library. mndD was subcloned as a 2.0-kb SmaI fragment in pUC18, from which manganese-dependent extradiol dioxygenase activity was expressed at high levels in Escherichia coli. The mndD open reading frame was identified by comparison with the known N-terminal amino acid sequence of purified manganese-dependent 3,4-DHPA 2,3-dioxygenase. Fourteen of 18 amino acids conserved in members of the iron-dependent extradiol dioxygenase family are also conserved in the manganese-dependent 3,4-DHPA 2,3-dioxygenase (MndD). Thus, MndD belongs to the extradiol family of dioxygenases and may share a common ancestry with the iron-dependent extradiol dioxygenases. We propose the revised consensus primary sequence (G,T,N,R)X(H,A)XXXXXXX(L,I,V,M,F)YXX(D,E,T,N,A)PX(G,P) X(2,3)E for this family. (Numbers in brackets indicate a gap of two or three residues at this point in the sequence.) The suggested common ancestry is also supported by sequence obtained from genes flanking mndD, which share significant sequence identity with xylJ and xylG from Pseudomonas putida.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2005

Molecular characterization of the gallate dioxygenase from Pseudomonas putida KT2440. The prototype of a new subgroup of extradiol dioxygenases.

In this work we have characterized the galA gene product from Pseudomonas putida KT2440, a ring-cleavage dioxygenase that acts specifically on gallate to produce 4-oxalomesaconate. The protein is a trimer composed by three identical subunits of 47.6 kDa (419 amino acids) that uses Fe2+ as the main cofactor. The gallate dioxygenase showed maximum activity at pH 7.0, and the Km and Vmax values for gallate were 144 microM and 53.2 micromol/min/mg of protein, respectively. A phylogenetic study suggests that the gallate dioxygenase from P. putida KT2440 is the prototype of a new subgroup of type II extradiol dioxygenases that share a common ancestor with protocatechuate 4,5-dioxygenases and whose two-domain architecture might have evolved from the fusion of the large and small subunits of the latter. A three-dimensional model for the N-terminal domain (residues 1-281) and C-terminal domain (residues 294-420) of the gallate dioxygenase from P. putida KT2440 was generated by comparison with the crystal structures of the large (LigB) and small (LigA) subunits of the protocatechuate 4,5-dioxygenase from Sphingomonas paucimobilis SYK-6. The expression of the galA gene was specifically induced when P. putida KT2440 cells grew in the presence of gallate. A P. putida KT2440 galA mutant strain was unable to use gallate as the sole carbon source and it did not show gallate dioxygenase activity, suggesting that the GalA protein is the only dioxygenase involved in gallate cleavage in this bacterium. This work points to the existence of a new pathway that is devoted to the catabolism of gallic acid and that remained unknown in the paradigmatic P. putida KT2440 strain.

Amino Acid Sequence↗

PubMed2003

Expression of chlorocatechol 1,2-dioxygenase and chlorocatechol 2,3-dioxygenase genes in chlorobenzene-contaminated subsurface samples.

In order to evaluate the in situ degradative capabilities of microorganisms in an underground reactor facility housing two flowthrough columns filled with aquifer soil, we examined the distribution and phylogeny of gene transcripts encoding enzymes capable of catalyzing the cleavage of the chlorinated aromatic ring during transformation of the main pollutant, chlorobenzene. Initial biostimulation of the autochthonous bacteria in the originally anaerobic reactor columns was achieved by injecting nitrate and oxygen in the form of H(2)O(2). Two broad-range primer pairs were used for reverse transcriptase PCR (RT-PCR) of partial subunit genes of chlorocatechol 1,2-dioxygenase and catechol 2,3-dioxygenase from RNA directly extracted from different groundwater and aquifer samples. Samples retrieved from the lowermost sections of the reactor columns, which were operated in upflow mode, were positive for the presence of chlorocatechol 1,2-dioxygenase and catechol 2,3-dioxygenase mRNA. On the other hand, chlorocatechol 1,2-dioxygenase RT-PCR products were detected in a larger part of each reactor column, up to a zone 5.5 m above the bottom. Phylogenetic analyses of these chlorocatechol 1,2-dioxygenase sequences clearly separated them into two main clusters, one of which was closely affiliated with the broad-spectrum chlorocatechol 1,2-dioxygenase from Pseudomonas chlororaphis RW71. Analysis of sequences obtained from RT-PCR products amplified with catechol 2,3-dioxygenase primers revealed that their closest relative was the chlorocatechol 2,3-dioxygenase gene cbzE from Pseudomonas putida GJ31 (A. E. Mars, J. Kingma, S. R. Kaschabek, W. Reineke, and D. B. Janssen, J. Bacteriol. 181:1309-1318, 1999), with sequence similarities between 97.8 and 99.0%.

Bacteria↗

PubMed1991

Overproduction, purification, and characterization of chlorocatechol dioxygenase, a non-heme iron dioxygenase with broad substrate tolerance.

We show here that purified chlorocatechol dioxygenase from Pseudomonas putida is able to oxygenate a wide range of substituted catechols with turnover numbers ranging from 2 to 29 s-1. This enzyme efficiently cleaves substituted catechols bearing electron-donating or multiple electron-withdrawing groups in an intradiol manner with kcat/KM values between 0.2 x 10(7) and 1.4 x 10(7) M-1 s-1. These unique catalytic properties prompted a comparison with the related but highly specific enzymes catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase. The chlorocatechol dioxygenase gene (clcA) from the Pseudomonas plasmid pAC27 was subcloned into the expression vector pKK223-3, allowing production of chlorocatechol dioxygenase to approximately 7-8% of total cellular protein. An average of 4 mg of purified enzyme has been obtained per gram of wet cells. Protein and iron analyses indicate an iron stoichiometry of 1 iron/57.5-kDa homodimer, alpha 2Fe. The electronic absorption spectrum contains a broad tyrosinate to iron charge transfer transition centered at 430 nm (epsilon = 3095 M-1 cm-1 based on iron concentration) which shifts to 490 nm (epsilon = 3380 M-1 cm-1) upon catechol binding. The resonance Raman spectrum of the native enzyme exhibits characteristic tyrosine ring vibrations. Electron paramagnetic resonance data for the resting enzyme (g = 4.25, 9.83) is consistent with high-spin iron (III) in a rhombic environment. This similarity between the spectroscopic properties of the Fe(III) centers in chlorocatechol dioxygenase and the more specific dioxygenases suggests a highly conserved catalytic site. We infer that the unique catalytic properties of chlorocatechol dioxygenase are due to other characteristics of its substrate binding pocket.

Catechol 1,2-Dioxygenase↗

PubMed1999

An archetypical extradiol-cleaving catecholic dioxygenase: the crystal structure of catechol 2,3-dioxygenase (metapyrocatechase) from Ppseudomonas putida mt-2.

BACKGROUND: Catechol dioxygenases catalyze the ring cleavage of catechol and its derivatives in either an intradiol or extradiol manner. These enzymes have a key role in the degradation of aromatic molecules in the environment by soil bacteria. Catechol 2, 3-dioxygenase catalyzes the incorporation of dioxygen into catechol and the extradiol ring cleavage to form 2-hydroxymuconate semialdehyde. Catechol 2,3-dioxygenase (metapyrocatechase, MPC) from Pseudomonas putida mt-2 was the first extradiol dioxygenase to be obtained in a pure form and has been studied extensively. The lack of an MPC structure has hampered the understanding of the general mechanism of extradiol dioxygenases. RESULTS: The three-dimensional structure of MPC has been determined at 2.8 A resolution by the multiple isomorphous replacement method. The enzyme is a homotetramer with each subunit folded into two similar domains. The structure of the MPC subunit resembles that of 2,3-dihydroxybiphenyl 1,2-dioxygenase, although there is low amino acid sequence identity between these enzymes. The active-site structure reveals a distorted tetrahedral Fe(II) site with three endogenous ligands (His153, His214 and Glu265), and an additional molecule that is most probably acetone. CONCLUSIONS: The present structure of MPC, combined with those of two 2,3-dihydroxybiphenyl 1,2-dioxygenases, reveals a conserved core region of the active site comprising three Fe(II) ligands (His153, His214 and Glu265), one tyrosine (Tyr255) and two histidine (His199 and His246) residues. The results suggest that extradiol dioxygenases employ a common mechanism to recognize the catechol ring moiety of various substrates and to activate dioxygen. One of the conserved histidine residues (His199) seems to have important roles in the catalytic cycle.

Amino Acid Sequence↗

PubMed1994

3-Sulphocatechol 2,3-dioxygenase and other dioxygenases (EC 1.13.11.2 and EC 1.14.12.-) in the degradative pathways of 2-aminobenzenesulphonic, benzenesulphonic and 4-toluenesulphonic acids in Alcaligenes sp. strain O-1.

Alcaligenes sp. strain O-1 utilizes three sulphonated aromatic compounds as sole sources of carbon and energy for growth in minimal salts medium-benzenesulphonate (BS), 4-toluenesulphonate (TS) and 2-aminobenzenesulphonate (2AS). The degradative pathway(s) in 2AS-grown cells are initiated with membrane transport, NADH-dependent dioxygenation and meta ring cleavage. The specific activity of the NADH-dependent dioxygenation(s) varied with the growth phase and was maximal near the end of exponential growth for each growth substrate. Cells were harvested at this point from BS-, TS- and 2AS-salts medium. Cells grown with each sulphonated substrate could oxygenate all three compounds, but only 2AS-grown cells consumed 2 mol O2 per mol 2AS or BS or TS. BS- and TS-grown cells consumed 2 mol O2 per mol BS or TS but failed to oxygenate the product of oxygenation of 2AS, 3-sulphocatechol (3SC). These observations were repeated with cell extracts and we concluded that there were two sets of desulphonative pathways in the organism, one for 2AS and one for BS and TS. We confirmed this hypothesis by separating the degradative enzymes from 2AS-, BS- or TS-grown cells. A 2AS dioxygenase system and a 3SC-2,3-dioxygenase (3SC23O) were detected in 2AS-grown cells only. In both BS- and TS-grown cells a dioxygenase system for BS and TS was observed as well as a principal catechol 2,3-dioxygenase (C23O-III), neither of which was present in 2AS-grown cells. The 3SC23O was purified to near homogeneity, found to be monomeric (M(r) 42,000), and to catalyse 2,3-dioxygenation to a product that decayed spontaneously to sulphite and 2-hydroxymuconate. The 2AS dioxygenase system could cause not only deamination of 2AS but also desulphonation of BS and TS. The BS dioxygenase could desulphonate BS and apparently either desulphonate or deaminate 2AS. Strain O-1 thus seems to contain two putative, independently regulated operons involving oxygenation and spontaneous desulphonation(s). One operon encodes at least the 2AS dioxygenase system and 3SC23O whereas the other encodes at least the BS/TS dioxygenase system and C23O-III.

Alcaligenes↗

PubMed1996

Homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum. A dioxygenase with catalase activity.

Homoprotocatechuate 2,3-dioxygenase (2,3-HPCD) cleaves the aromatic ring of its substrate with insertion of both atoms of oxygen from O2 to form alpha-hydroxy- delta-carboxymethyl cis-muconic semialdehyde. The enzyme has been purified from the Gram-positive bacterium Brevibacterium fuscum and characterized. The enzyme appears to have a range of quaternary structures with predominant components of alpha4 and alpha6 (alpha subunit Mr = 42500 +/- 1500) and binds approximately 1 Fe(II)/subunit. Although the substrate Km values are similar to those of other Fe(II) ring cleaving dioxygenases, the turnover number is lower by 90-97%, and the enzyme exhibits much higher stability to metal chelators and H2O2. The stability to H2O2 is shown to derive from an endogenous catalase activity of 2,3-HPCD (stoichiometry: 2 H2O2 --> 2 H2O + O2) that is novel for dioxygenases. H2O2 is a mixed-type inhibitor of the dioxygenase activity, suggesting that dioxygenase and catalase activities are both catalyzed by the enzyme, but at distinguishable sites. In contrast, catecholic substrates, including homoprotocatechuate and p-nitrocatechol, are nonessential activators of the catalase activity. The plot of 1/vi of catalase activity versus 1/[H2O2] is parabolic in the absence of catecholic substrates and linear in their presence, indicating that these reactions proceed by different mechanisms. A mechanism for catalase activity is proposed in which 2 H2O2 molecules bind simultaneously to the iron to account for the observed parabolic kinetic plot. Electron transfer between the peroxides mediated by the iron would yield 2 H2O and O2. Catecholic substrates are proposed to modify this reaction by excluding one H2O2 from the Fe(II), thereby causing the kinetic plots to appear linear. Electron donation by the catecholic substrates would facilitate O O bond cleavage of H2O2, but outer sphere electron transfer from a second H2O2 in another step would be necessary to complete the reaction. p-Nitrocatechol is shown to bind differently to 2,3-HPCD than to other Fe(II) ring cleavage dioxygenases. Possible explanations for this observation are considered in the context of the proposed catalase and normal dioxygenase mechanisms which may also have bearing on the unique catalase activity and low dioxygenase turnover number of the enzyme.

Amino Acids↗

PubMed1996

Catechol dioxygenases from Escherichia coli (MhpB) and Alcaligenes eutrophus (MpcI): sequence analysis and biochemical properties of a third family of extradiol dioxygenases.

The nucleotide sequence of the Escherichia coli mhpB gene, encoding 2,3-dihydroxyphenylpropionate 1,2-dioxygenase, was determined by sequencing of a 3.1-kb fragment of DNA from Kohara phage 139. The inferred amino acid sequence showed 58% sequence identity with the sequence of an extradiol dioxygenase, MpcI, from Alcaligenes eutrophus and 10 to 20% sequence identity with protocatechuate 4,5-dioxygenase from Pseudomonas paucimobilis, with 3,4-dihydroxyphenylacetate 2,3-dioxygenase from E. coli, and with human 3-hydroxyanthranilate dioxygenase. Sequence similarity between the N- and C-terminal halves of this new family of dioxygenases was detected, with conserved histidine residues in the N-terminal domain. A model is proposed to account for the relationship between this family of enzymes and other extradiol dioxygenases. The A. eutrophus MpcI enzyme was expressed in E. coli, purified, and characterized as a protein with a subunit size of 33.8 kDa. Purified MhpB and MpcI showed similar substrate specificities for a range of 3-substituted catechols, and evidence for essential histidine and cysteine residues in both enzymes was obtained.

Alcaligenes↗

PubMed1996

Studies on the isopropylbenzene 2,3-dioxygenase and the 3-isopropylcatechol 2,3-dioxygenase genes encoded by the linear plasmid of Rhodococcus erythropolis BD2.

The enzymes responsible for the degradation of isopropylbenzene (IPB) and co-oxidation of trichloroethene (TCE) by Rhodococcus erythropolis BD2 are encoded by the linear plasmid pBD2. Fragments containing IPB catabolic genes were cloned from pBD2 and the nucleotide sequence was determined. By means of database searches and expression of the cloned genes in recombinant strains, we identified five clustered genes, ipbA1A2A3A4C, which encode the three components of the IPB 2,3-dioxygenase system, reductaseIPB (ipbA4), ferredoxinIPB (ipbA3) and the two subunits of the terminal dioxygenase (ipbA1A2), as well as the 3-isopropylcatechol (IPC) 2,3-dioxygenase (ipbC). The protein sequences deduced from the ipbA1A2A3A4C gene cluster exhibited significant homology with the corresponding proteins of analogous degradative pathways in Gram-negative and Gram-positive bacteria, but the gene order differed from most of them. IPB 2,3-dioxygenase and 3-IPC 2,3-dioxygenase could both be expressed in Escherichia coli, but the IPB 2,3-dioxygenase activities were too low to be detected by polarographic and TCE degradative means. However, inhibitor studies with the R. erythropolis BD2 wild-type are in accordance with the involvement of the IPB 2,3-dioxygenase in TCE oxidation.

Amino Acid Sequence↗

PubMed1996

2,4-Dinitrotoluene dioxygenase from Burkholderia sp. strain DNT: similarity to naphthalene dioxygenase.

2,4-Dinitrotoluene (DNT) dioxygenase from Burkholderia sp. strain DNT catalyzes the initial oxidation of DNT to form 4-methyl-5-nitrocatechol (MNC) and nitrite. The displacement of the aromatic nitro group by dioxygenases has only recently been described, and nothing is known about the evolutionary origin of the enzyme systems that catalyze these reactions. We have shown previously that the gene encoding DNT dioxygenase is localized on a degradative plasmid within a 6.8-kb NsiI DNA fragment (W.-C. Suen and J. C. Spain, J. Bacteriol. 175:1831-1837, 1993). We describe here the sequence analysis and the substrate range of the enzyme system encoded by this fragment. Five open reading frames were identified, four of which have a high degree of similarity (59 to 78% identity) to the components of naphthalene dioxygenase (NDO) from Pseudomonas strains. The conserved amino acid residues within NDO that are involved in cofactor binding were also identified in the gene encoding DNT dioxygenase. An Escherichia coli clone that expressed DNT dioxygenase converted DNT to MNC and also converted naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. In contrast, the E. coli clone that expressed NDO did not oxidize DNT. Furthermore, the enzyme systems exhibit similar broad substrate specificities and can oxidize such compounds as indole, indan, indene, phenetole, and acenaphthene. These results suggest that DNT dioxygenase and the NDO enzyme system share a common ancestor.

Amino Acid Sequence↗

PubMed1984

Inhibition of indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase by beta-carboline and indole derivatives.

beta-Carboline derivatives inhibited both indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase activities from various sources. Among them, norharman is most potent for both enzymes from mammalian sources. Kinetic studies revealed that norharman is uncompetitive (Ki = 0.12 mM) with L-tryptophan for rabbit intestinal indoleamine 2,3-dioxygenase, and linearly competitive (Ki = 0.29 mM) with L-tryptophan for mouse liver tryptophan 2,3-dioxygenase. In addition, some beta-carbolines selectively inhibited one enzyme or the other. Pseudomonad tryptophan 2,3-dioxygenase was inhibited by a different spectrum of beta-carbolines. Such a selective inhibition by the structure of substrate analogs is more evident by the use of indole derivatives. Indole-3-acetamide, indole-3-acetonitrile and indole-3-acrylic acid exhibited a potent inhibition for mammalian tryptophan 2,3-dioxygenase, while they moderately inhibited the pseudomonad enzyme. However, they showed no inhibition for indoleamine 2,3-dioxygenase. These results suggest the difference of the structures of the active sites among these enzymes from various sources.

Animals↗

PubMed1999

Conversion of 3-chlorocatechol by various catechol 2,3-dioxygenases and sequence analysis of the chlorocatechol dioxygenase region of Pseudomonas putida GJ31.

Pseudomonas putida GJ31 contains an unusual catechol 2,3-dioxygenase that converts 3-chlorocatechol and 3-methylcatechol, which enables the organism to use both chloroaromatics and methylaromatics for growth. A 3.1-kb region of genomic DNA of strain GJ31 containing the gene for this chlorocatechol 2,3-dioxygenase (cbzE) was cloned and sequenced. The cbzE gene appeared to be plasmid localized and was found in a region that also harbors genes encoding a transposase, a ferredoxin that was homologous to XylT, an open reading frame with similarity to a protein of a meta-cleavage pathway with unknown function, and a 2-hydroxymuconic semialdehyde dehydrogenase. CbzE was most similar to catechol 2,3-dioxygenases of the 2.C subfamily of type 1 extradiol dioxygenases (L. D. Eltis and J. T. Bolin, J. Bacteriol. 178:5930-5937, 1996). The substrate range and turnover capacity with 3-chlorocatechol were determined for CbzE and four related catechol 2,3-dioxygenases. The results showed that CbzE was the only enzyme that could productively convert 3-chlorocatechol. Besides, CbzE was less susceptible to inactivation by methylated catechols. Hybrid enzymes that were made of CzbE and the catechol 2, 3-dioxygenase of P. putida UCC2 (TdnC) showed that the resistance of CbzE to suicide inactivation and its substrate specificity were mainly determined by the C-terminal region of the protein.

Amino Acid Sequence↗

PubMed1999

Crystal structure of an aromatic ring opening dioxygenase LigAB, a protocatechuate 4,5-dioxygenase, under aerobic conditions.

BACKGROUND: Sphingomonas paucimobilis SYK-6 utilizes an extradiol-type catecholic dioxygenase, the LigAB enzyme (a protocatechuate 4,5-dioxygenase), to oxidize protocatechuate (or 3,4-dihydroxybenzoic acid, PCA). The enzyme belongs to the family of class III extradiol-type catecholic dioxygenases catalyzing the ring-opening reaction of protocatechuate and related compounds. The primary structure of LigAB suggests that the enzyme has no evolutionary relationship with the family of class II extradiol-type catecholic dioxygenases. Both the class II and class III enzymes utilize a non-heme ferrous center for adding dioxygen to the substrate. By elucidating the structure of LigAB, we aimed to provide a structural basis for discussing the function of class III enzymes. RESULTS: The crystal structure of substrate-free LigAB was solved at 2.2 A resolution. The molecule is an alpha2beta2 tetramer. The active site contains a non-heme iron coordinated by His12, His61, Glu242, and a water molecule located in a deep cleft of the beta subunit, which is covered by the alpha subunit. Because of the apparent oxidation of the Fe ion into the nonphysiological Fe(III) state, we could also solve the structure of LigAB complexed with a substrate, PCA. The iron coordination sphere in this complex is a distorted tetragonal bipyramid with one ligand missing, which is presumed to be the O2-binding site. CONCLUSIONS: The structure of LigAB is completely different from those of the class II extradiol-type dioxygenases exemplified by the BphC enzyme, a 2,3-dihydroxybiphenyl 1,2-dioxygenase from a Pseudomonas species. Thus, as already implicated by the primary structures, no evolutionary relationship exists between the class II and III enzymes. However, the two classes of enzymes share many geometrical characteristics with respect to the nature of the iron coordination sphere and the position of a putative catalytic base, strongly suggesting a common catalytic mechanism.

Amino Acid Sequence↗

PubMed2005

Is it true dioxygenase or classic autoxidation catalysis? Re-investigation of a claimed dioxygenase catalyst based on a Ru(2)-incorporated, polyoxometalate precatalyst.

A 1997 Nature paper reported that a novel Ru(2)-incorporated sandwich-type polyoxometalate, {[WZnRu(III)(2)(OH)(H(2)O)](ZnW(9)O(34))(2)}(11)(-), is an all-inorganic dioxygenase catalyst for the hydroxylation of adamantane and the epoxidation of alkenes using molecular oxygen. Specifically, it was reported that the above Ru(2)-containing polyoxometalate catalyzes the following reaction by a non-radical-chain, dioxygenase mechanism: 2RH + O(2) --> 2ROH (R = adamantane). A re-investigation of the above claim has been performed, resulting in the following findings: (1) iodometric analysis detects trace peroxides (0.5% relative to adamantane), the products of free-radical-chain autoxidation, at the end of the adamantane hydroxylation reaction; (2) a non-dioxygenase product, H(2)(18)O, is observed at the end of an adamantane hydroxylation reaction performed using (18)O(2); (3) kinetic studies reveal a fractional rate law consistent with a classic radical-chain reaction; (4) a non-dioxygenase approximately 1:1 adamantane products/O(2) stoichiometry is observed in our hands (instead of the claimed 2:1 adamantane/O(2) dioxygenase stoichiometry); (5) adamantane hydroxylation is initiated by the free radical initiator, AIBN (2,2'-azobisisobutyronitrile), or the organic hydroperoxide, t-BuOOH; (6) four radical scavengers completely inhibit the reaction; and (7) {[WZnRu(III)(2)(OH)(H(2)O)](ZnW(9)O(34))(2)}(11)(-) is found to be an effective catalyst for cyclohexene free-radical-chain autoxidation. The above results are consistent with and strongly supportive of a free-radical-chain mechanism, not the previously claimed dioxygenase pathway.

Catalysis↗