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Catechol 1,2 dioxygenase is a versatile enzyme with several potential applications. However, due to its low thermostability, its industrial potential is not being met. In this study, the thermostability of a mesophilic catechol 1,2 dioxygenase from the species Rhodococcus opacus was enhanced via the introduction of disulphide bonds into its structure. Engineered designs (56) were obtained using computational prediction applications, with a set of hypothesized selection criteria narrowing the list to 9. Following recombinant production and purification, several of the designs demonstrated substantially improved protein thermostability. Notably, variant K96C-D278C yielded improvements including a 4.6°C increase in T 50 , a 725% increase in half-life, a 5.5°C increase in T m , and a >10-fold increase in total turnover number compared to wild type. Stacking of best designs was not productive. Overall, current state-of-the-art prediction algorithms were effective for design of disulfide-thermostabilized catechol 1,2 dioxygenase., (© 2024 National Research Council Canada and The Authors. Biotechnology and Bioengineering by Wiley Periodicals LLC. Reproduced with the permission of the Minister of Innovation, Science, and Economic Development.)

Microbial remediation has become one of the promising ways to eliminate polycyclic aromatic hydrocarbons (PAHs) pollution due to its efficient enzyme metabolism system. Catechol 1,2-dioxygenase (C12O) is a crucial rate-limiting enzyme in the degradation pathway of PAHs in Achromobacter xylosoxidans DN002 that opens the benzene ring through the ortho-cleavage pathway. However, little attention has been given to explore the interaction mechanism of relevant enzyme-substrate. This study aims to investigate the binding interaction between C12O of strain DN002 and catechol by means of a molecular biological approach combined with homology modeling, molecular docking, and multiple spectroscopies. The removal rate of catechol in the mutant strain of cat A deletion was only 12.03%, compared to the wild-type strain (54.21%). A Ramachandran plot of active site regions of the primary amino acid sequences in the native enzyme showed that 93.5% sequences were in the most favored regions on account of the results of homology modeling, while an additional 6.2% amino acid sequences were found in conditionally allowed regions, and 0.4% in generously allowed regions. The binding pocket of C12O with catechol was analyzed to obtain that the catalytic trimeric group of Tyr164-His224-His226 was proven to be great vital for the ring-opening reaction of catechol by molecular docking. In the native enzyme, binding complexes were spontaneously formed by hydrophobic interactions. Binding constants and thermodynamic potentials from fluorescence spectra indicated that catechol effectively quenched the intrinsic fluorescence of C12O in the C12O/catechol complex via conventional static and dynamic quenching mechanisms of C12O. The results of ultraviolet and visible (UV) spectra, synchronous fluorescence, and circular dichroism (CD) spectra revealed conspicuous changes in the local conformation, and site-directed mutagenesis confirmed the role of predicted key residues during catalysis, wherein His 226 had a significant effect on catechol utilization by C12O. This is the first report to reveal interactions of C12O with substrate from the molecular docking results, providing the mechanistic understanding of representative dioxygenases involved in aromatic compound degradation, and a solid foundation for further site modifications as well as strategies for the directed evolution of this enzyme., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The gene of catechol 1, 2-dioxygenase was identified and cloned from the genome of Oceanimonas marisflavi 102-Na3. The protein was expressed in Escherichia coli BL21 (DE3) and purified to homogeneity of a dimer with molecular mass of 69.2 kDa. The enzyme was highly stable in pH 6.0-9.5 and below 45 °C and exhibited the maximum activity at pH 8.0 and 30 °C. Being the first characterized intradiol dioxygenase from marine bacteria Oceanimonas sp., the enzyme showed catalytic activity for catechol, 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol and pyrogallol. For catechol, K m and V max were 11.2 μM and 13.4 U/mg of protein, respectively. The enzyme also showed resistance to most of the metal ions, surfactants and organic solvents, being a promising biocatalyst for biodegradation of aromatic compounds in complex environments., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The present study reports on the cloning, expression and characterization of catechol 1,2-dioxygenase (CAT) of bacterial strains isolated from dioxin-contaminated soils in Vietnam. Two isolated bacterial strains DF2 and DF4 were identified as Burkholderia cepacia based on their 16S rRNA sequences. Their genes coding CAT was amplified with a specific pair of primers. Recombinant CAT (rCAT) was expressed in E. coli M15 cells and its activity was confirmed by the detection of cis,cis-muconic acid, a product from catechol, by high-performance liquid chromatography (HPLC) analysis. The rCAT of DF4 had an optimal pH and temperature of 7 and 30°C, respectively. Metal ions, such as Zn 2+ and Mn 2+ , and surfactants, such as SDS, Tween 20 and Triton X100, strongly inhibited enzyme activity, while K + slightly increased the activity.

Catechol 1,2-dioxygenases catalyze catechol ring-opening, a critical step in the degradation of aromatic compounds. Cupriavidus campinensis BJ71, an efficient 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterial strain, was previously isolated from an environment contaminated with 2,4-D. In this study, catA encoding a catechol 1,2-dioxygenase was cloned from the BJ71 strain. The gene was 939 bp long and encoded a polypeptide of 312 amino acids with a molecular weight of 34 kDa. To investigate its enzymatic characteristics, CatA was heterologously expressed in Escherichia coli . Optimal reaction conditions for the pure enzyme were 35 °C and pH 8.0. The enzyme remained stable within a range of 25 °C-45 °C and pH 6.0-9.0, thus indicating that CatA has wide temperature and pH adaptability. After incubation at 45 °C, the enzyme activity of CatA decreased to 37.12%, but its activity was not affected by incubation at pH 9.0. The pure enzyme was able to use catechol, 4-methyl-catechol and 4-chlorocatechol as substrates. Enzyme kinetic parameters K m and V max were 39.97 µM and 10.68 U/mg, respectively. This is the first report of the cloning of a gene encoding a catechol 1,2-dioxygenase from a 2,4-D-degrading bacterial strain.

Catechol dioxygenases in microorganisms cleave catechol into cis - cis -muconic acid or 2-hydroxymuconic semialdehyde via the ortho - or meta -pathways, respectively. The aim of this study was to purify, characterize, and predict the template-based three-dimensional structure of catechol 1,2-dioxygenase (C12O) from indigenous Pseudomonas chlororaphis strain UFB2 ( Pc UFB2). Preliminary studies showed that Pc UFB2 could degrade 40 ppm of 2,4-dichlorophenol (2,4-DCP). The crude cell extract showed 10.34 U/mL of C12O activity with a specific activity of 2.23 U/mg of protein. A 35 kDa protein was purified to 1.5-fold with total yield of 13.02% by applying anion exchange and gel filtration chromatography. The enzyme was optimally active at pH 7.5 and a temperature of 30 °C. The Lineweaver⁻Burk plot showed the v max and K m values of 16.67 µM/min and 35.76 µM, respectively. ES-MS spectra of tryptic digested SDS-PAGE band and bioinformatics studies revealed that C12O shared 81% homology with homogentisate 1,2-dioxygenase reported in other Pseudomonas chlororaphis strains. The characterization and optimization of C12O activity can assist in understanding the 2,4-DCP metabolic pathway in Pc UFB2 and its possible application in bioremediation strategies.

The sequence cato encoding catechol 1,2-dioxygenase from Candida tropicalis JH8 was cloned, sequenced, and expressed in Escherichia coli. The sequence cato contained an ORF of 858 bp encoding a polypeptide of 285 amino acid residues. The recombinant catechol 1,2-dioxygenase exists as a homodimer structure with a subunit molecular mass of 32 KD. Recombinant catechol 1,2-dioxygenase was unstable below pH 5.0 and stable from pH 7.0 to 9.0; its optimum pH was at 7.5. The optimum temperature for the enzyme was 30°C, and it possessed a thermophilic activity within a broad temperature range. Under the optimal conditions with catechol as substrate, the Km and Vmax of recombinant catechol 1,2-dioxygenase were 9.2 µM and 0.987 µM/min, respectively. This is the first article presenting cloning and expressing in E. coli of catechol 1,2-dioxygenase from C. tropicalis and characterization of the recombinant catechol 1,2-dioxygenase.

This study intends to purify and characterize catechol 1,2-dioxygenase (C1,2O) of phenol-degrading Acinetobacter sp. Y64 and of E. coli transformant. Acinetobacter sp. Y64 was capable of degrading 1000 mg/L of phenol within 14 ± 2 h at 30 °C, 160 rpm and pH of 7. One C1,2O of 36 kDa was purified using ammonium sulphate precipitation and Hitrap QFF column chromatograph with 49% recovery and a 10.6-fold increase in purity. Purified Y64 C1,2O had temperature and pH optimum at 37 °C and pH 7.7 respectively with the Michaelis constant of 17.53 µM and the maximal velocity of 1.95 U/mg, respectively. The presence of Fe(3+) or Fe(2+) enhanced the activity of Y64 C1,2O while other compounds such as Ca(2+), and EDTA had an inhibitory effect. 80% of C1,2O activity remained using 4-nitrocatechol as substrate while 2% remained using 3-methylcatechol compared with that using catechol. Y64 catA gene encoding C1,2O was amplified using PCR cloned into pET22b vector and expressed in Escherichia coli BL21 DE3 (pLysS) after transformation. Purified and cloned Y64 C1,2O show no significant differences in the biochemical properties. The phylogenetic tree based on the protein sequences indicates that these C1,2Os possess a common ancestry.

A few mononuclear iron(iii) complexes of the type [Fe(L)Cl2]Cl , where L is a tetradentate tripodal 4N ligand such as N,N-dimethyl-N',N'-bis(pyrid-2-ylmethyl)ethane-1,2-diamine (), N,N-diethyl-N',N'-bis(pyrid-2-ylmethyl)ethane-1,2-diamine (), N,N-dimethyl-N',N'-bis-(6-methylpyrid-2-ylmethyl)ethane-1,2-diamine (), N,N-dimethyl-N'-(pyrid-2-ylmethyl)-N'-(1-methyl-1H-imidazol-2-ylmethyl)ethane-1,2-diamine (), N,N-dimethyl-N',N'-bis(1-methyl-1H-imidazol-2-ylmethyl)ethane-1,2-diamine () and N,N-dimethyl-N',N'-bis(quinolin-2-ylmethyl)ethane-1,2-diamine (), have been isolated and characterized by CHN analysis, UV-Visible spectroscopy and electrochemical methods. The complex cation [Fe(H)Cl3](+) possesses a distorted octahedral geometry in which iron is coordinated by the monoprotonated 4N ligand in a tridentate fashion and the remaining three sites of the octahedron are occupied by chloride ions. The DFT optimized octahedral geometries of , and contain iron(iii) with a high-spin (S = 5/2) ground state. The catecholate adducts [Fe(L)(DBC)](+), where H2DBC is 3,5-di-tert-butylcatechol, of all the complexes have been generated in situ in acetonitrile solution and their spectral and redox properties and dioxygenase activities have been studied. The DFT optimized geometries of the catecholate adducts [Fe()(DBC)](+), [Fe()(DBC)](+) and [Fe()(DBC)](+) have also been generated to illustrate the ability of the complexes to cleave H2DBC in the presence of molecular oxygen to afford varying amounts of intra- (I) and extradiol (E) cleavage products. The extradiol to intradiol product selectivity (E/I, 0.1-2.0) depends upon the asymmetry in bidentate coordination of catecholate, as determined by the stereoelectronic properties of the ligand donor functionalities. While the higher E/I value obtained for [Fe()(DBC)](+) is on account of the steric hindrance of the quinolyl moiety to coordination the lower value observed for [Fe()(DBC)](+) and [Fe()(DBC)](+) is on account of the electron-releasing effect of the N-methylimidazolyl moiety. Based on the data obtained it is proposed that the detachment of the -NMe2 group from the coordination sphere in the semiquinone intermediate is followed for dioxygen binding and activation to yield the extradiol cleavage product.

The enzymatic decomposition of 4-chlorophenol metabolites using an immobilized biocatalyst was investigated in this study. Catechol 1,2-dioxygenase for ortho ring cleavage obtained via cloning of the corresponding gene cphA-I from Arthrobacter chlorophenolicus A6 was overexpressed and purified. It was found that the cphA-I enzyme could catalyze the degradation of catechol, 4-chlorocatechol, and 3-methylcatechol. The expressed enzyme was immobilized onto a natural enzyme support, fulvic acid-activated montmorillonite. The immobilization yield was as high as 63%, and the immobilized enzyme maintained high substrate utilization activity, with only a 15-24% reduction in the specific activity. Kinetic analysis demonstrated marginal differences in νmax and KM values for the free and immobilized enzymes, indicating that inactivation of the immobilized enzyme was minimal. The immobilized enzyme exhibited notably increased stability against changes in the surrounding environment (temperature, pH, and ionic strength). Our results provide useful information for the effective enzymatic biochemical treatment of hazardous organic compounds., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

This is the first report of a catechol 1,2-dioxygenase from Stenotrophomonas maltophilia strain KB2 with high activity against catechol and its methyl derivatives. This enzyme was maximally active at pH 8.0 and 40 °C and the half-life of the enzyme at this temperature was 3 h. Kinetic studies showed that the value of K m and V max was 12.8 μM and 1,218.8 U/mg of protein, respectively. During our studies on kinetic properties of the catechol 1,2-dioxygenase we observed substrate inhibition at >80 μM. The nucleotide sequence of the gene encoding the S. maltophilia strain KB2 catechol 1,2-dioxygenase has high identity with other catA genes from members of the genus Pseudomonas. The deduced 314-residue sequence of the enzyme corresponds to a protein of molecular mass 34.5 kDa. This enzyme was inhibited by competitive inhibitors (phenol derivatives) only by ca. 30 %. High tolerance against condition changes is desirable in industrial processes. Our data suggest that this enzyme could be of use as a tool in production of cis,cis-muconic acid and its derivatives.

In vitro degradation of pyrene was studied in MSM by three bacterial strains individually, designated as BP10, NJ2 and P2. Among these strains, NJ2 was the highest degrader (60%) of pyrene, followed by BP10 (44%) and the least was P2 (42%) in MSM with pyrene (50 μg ml(-1)) in 8 days. During pyrene degradation, catechol 1,2 dioxygenase (C12O) activity was induced by 13 folds in BP10 and 17 folds in P2 as compared to catechol 2,3 dioxygenase (C23O). However, in NJ2, C23O activity was augmented 1.3 times more than C12O. This clearly indicated that C12O played a major role in pyrene degradation by BP10 and P2, while in NJ2, C23O contributed more to degradation process than C12O. Molecular weight of highly inducible C12O was determined as ~64 kDa by size exclusion chromatography and as ~32 kDa on denaturing SDS PAGE in BP10 which indicated dimeric nature of the enzyme., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Six dichloroiron(III) complexes of 1,3-bis(2'-arylimino)isoindoline (BAIH) with various N-donor aryl groups have been characterized by spectroscopy (infrared, UV-vis), electrochemistry (cyclic voltammetry), microanalysis, and in two cases X-ray crystallography. The structurally characterized Fe(III)Cl(2)(L(n)) complexes (n = 3, L(3) = 1,3-bis(2'-thiazolylimino)isoindoline and n = 5, L(5) = 1,3-bis(4-methyl-2'-piridylimino)isoindoline) are five-coordinate, trigonal bipyramidal with the isoindoline ligands occupying the two axial and one equatorial positions meridionally. These compounds served as precursors for catechol dioxygenase models that were formed in solution upon addition of 3,5-di-tert-butylcatechol (H(2)DBC) and excess triethylamine. These adducts react with dioxygen in N,N-dimethylformamide, and the analysis of the products by chromatography and mass spectrometry showed high intradiol over extradiol selectivity (the intradiol/extradiol product ratios varied between 46.5 and 6.5). Kinetic measurements were performed by following the change in the intensity of the catecholate to iron ligand-to-metal charge transfer (LMCT) band, the energy of which is influenced by the isoindolinate-ligand (827-960 nm). In combination with electrochemical investigations the kinetic studies revealed an inverse trend between reaction rates and oxidation potentials associated with the coordinated DBC(2-). On the basis of these results, a substrate activation mechanism is suggested for this system in which the geometry of the peroxide-bridged intermediate may be of key importance in regioselectivity.

Intradiol dioxygenase are iron-containing enzymes involved in the bacterial degradation of natural and xenobiotic aromatic compounds. The wild-type and mutants forms of catechol 1,2-dioxygenase Iso B from Acinetobacter radioresistens LMG S13 have been investigated in order to get an insight on the structure-function relationships within this system. 4K CW-EPR spectroscopy highlighted different oxygen binding properties of some mutants with respect to the wild-type enzyme, suggesting that a fine tuning of the substrate-binding determinants in the active site pocket may indirectly result in variations of the iron reactivity. A thermostability investigation by optical spectroscopy, that reports on the state of the metal center, showed that the structural stability is more influenced by the type rather than by the position of the mutation. Finally, the influence of pH and temperature on the catalytic activity was monitored and discussed in terms of perturbations induced on the tertiary contact network of the enzyme.

An enzyme-magnetic nanoparticle conjugate is prepared via conjugation of Ni(2+) ions onto the surface of magnetic nanoparticles to interact with a six histidine-tagged enzyme. The catalytic properties and enzyme rigidification of the conjugates are more stable at high concentrations of aromatic hydrocarbons.

Intradiol-cleaving catechol 1,2 dioxygenases are Fe(III) dependent enzymes that act on catechol and substituted catechols, including chlorocatechols pollutants, by inserting molecular oxygen in the aromatic ring. Members of this class are the object of intense biochemical investigations aimed at the understanding of their catalytic mechanism, particularly for designing mutants with selected catalytic properties. We report here an in depth investigation of catechol 1,2 dioxygenase IsoB from Acinetobacter radioresistens LMG S13 and its A72G and L69A mutants. By applying a multidisciplinary approach that includes high resolution X-rays crystallography, mass spectrometry and single crystal microspectrophotometry, we characterised the phospholipid bound to the enzyme and provided a structural framework to understand the inversion of substrate specificity showed by the mutants. Our results might be of help for the rational design of enzyme mutants showing a biotechnologically relevant substrate specificity, particularly to be used in bioremediation. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We theoretically investigated the ligand-to-metal charge-transfer (LMCT) excitation of the native iron(III)-dependent catechol dioxygenase and its functional model complexes with multistate complete active space second-order perturbation theory (MS-CASPT2) because the LMCT (catecholate-to-iron(III) charge-transfer) excitation energy is believed to relate to the reactivity of the native enzyme and its functional model complexes. The ground state calculated by the MS-CASPT2 method mainly consists of the iron(III)-catecholate electron configuration and moderately of the iron(II)-semiquinonate electron configuration for both of the enzyme active centers and the model complexes when the active center exists in the protein environment and the model complexes exist in the solution. However, the ground-state wave function mainly consists of the iron(II)-semiquinonate electron configuration for both the enzyme active site without a protein environment and the model complexes in vacuo. These results clearly show that the protein environment and solvent play important roles to determine the electronic structure of the catecholatoiron(III) complex. The LMCT excitation energy clearly relates to the weight of the iron(III)-catecholate configuration in the ground state. The reactivity and the LMCT excitation energy directly relate to the ionization potential of the catecholate (IP(CAT)) in the model complex. This is because the charge transfer from the catecholate moiety to the dioxygen molecule plays a key role to activate the dioxygen molecule. However, the reactivity of the native catechol dioxygenase is much larger than those of the model complexes, despite the similar IP(CAT) values, suggesting that other factors such as the coordinatively unsaturated iron(III) center of the native enzyme play a crucial role in the reactivity.

An investigation was carried out for in vitro degradation of fluoranthene by four bacterial strains (PSM6, PSM7, PSM10 and PSM11) isolated from the petroleum sludge. Although all the strains registered their growth in MSM with 100 ppm fluoranthene, PSM11 growth was better than other strains. Growth of bacterial strains invariably corresponded to their degradation potential of fluoranthene. After 168 h of incubation, 61% fluoranthene was degraded by PSM11, followed by PSM10 (48%) and PSM6 (42%) and the least was recorded in PSM7 (41%). Besides, 11% loss in fluoranthene was attributed to abiotic factors. Thirty-eight times more activity of catechol 2,3-dioxygenase than catechol 1,2-dioxygenase showed that it played a significant role in fluoranthene degradation. Molecular weight of catechol 2,3-dioxygenase isolated from PSM11 was determined as ∼ 136 kDa by size exclusion chromatography and 34 kDa on denaturing SDS-PAGE, indicating tetrameric nature of the enzyme., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The aim of the present work was to investigate the influence of alkylhydroxybenzenes (AHBs) and tyrosol, which belong to cell differentiation factors d(1) group of autoregulators on properties of biodegradation enzymes, catechol 1,2-dioxygenase (Cat 1,2-DO) and methylcatechol 1,2-dioxygenase (MCat 1,2-DO) of Rhodococcus opacus 6a. AHBs were found to have a greater effect on MCat 1,2-DO than on Cat 1,2-DO. It was expressed by more pronounced changes in the activity of MCat 1,2-DO with unsubstituted catechol at different AHB concentrations and by increasing thermostability of MCat 1,2-DO compared to Cat 1,2-DO under the protective action of AHBs. The compound C(7)-AHB shifted the maximum of dioxygenase activities towards higher temperatures and increased their operation optimum. AHBs changed the specificity constant of dioxygenases by decreasing/increasing the K(m)/V(max) value. For example, the increase in the V(max) value of 3,6-dichlorocatechol oxidation by Cat 1,2-DO in the presence of C(7)-AHB was 300-fold higher compared to the same reaction without AHB. The influence of cell differentiation factors on the properties of dimeric enzymes has been shown for the first time. It gives an idea of how the specificity of enzymes can be changed in vivo when strains contact new substrates. The work has shown the possibility of modification of the properties of dimeric enzymes towards the extension of enzyme activity with difficulty converted substrates or in more extreme conditions, which may be important for biotechnological processes.

The iron(iii) complexes of the bis(phenolate) ligands 1,4-bis(2-hydroxy-4-methyl-benzyl)-1,4-diazepane H(2)(L1), 1,4-bis(2-hydroxy-4-nitrobenzyl)-1,4-diazepane H(2)(L2), 1,4-bis(2-hydroxy-3,5-dimethylbenzyl)-1,4-diazepane H(2)(L3) and 1,4-bis(2-hydroxy-3,5-di-tert-butylbenzyl)-1,4-diazepane H(2)(L4) have been isolated and studied as structural and functional models for 3,4-PCD enzymes. The complexes [Fe(L1)Cl] 1, [Fe(L2)(H(2)O)Cl] 2, [Fe(L3)Cl] 3 and [Fe(L4)Cl] 4 have been characterized using ESI-MS, elemental analysis, and absorption spectral and electrochemical methods. The single crystal X-ray structure of 3 contains the FeN(2)O(2)Cl chromophore with a novel square pyramidal (τ, 0.20) coordination geometry. The Fe-O-C bond angle (135.5°) and Fe-O bond length (1.855 Å) are very close to the Fe-O-C bond angles (133, 148°) and Fe-O(tyrosinate) bond distances (1.81, 1.91 Å) in 3,4-PCD enzyme. All the complexes exhibit two intense absorption bands in the ranges 335-383 and 493-541 nm, which are assigned respectively to phenolate (pπ) → Fe(iii) (dσ*) and phenolate (pπ) → Fe(iii) (dπ*) LMCT transitions. The Fe(iii)/Fe(ii) redox potentials of 1, 3 and 4 (E(1/2), -0.882--1.010 V) are more negative than that of 2 (E(1/2), -0.577 V) due to the presence of two electron-withdrawing p-nitrophenolate moieties in the latter enhancing the Lewis acidity of the iron(iii) center. Upon adding H(2)DBC pretreated with two equivalents of Et(3)N to the iron(iii) complexes, two catecholate-to-iron(iii) LMCT bands (656, ε, 1030; 515 nm, ε, 1330 M(-1) cm(-1)) are observed for 2; however, interestingly, an intense catecholate-to-iron(iii) LMCT band (530-541 nm) is observed for 1, 3 and 4 apart from a high intensity band in the range 451-462 nm. The adducts [Fe(L)(DBC)](-) generated from 1-4in situ in DMF/Et(3)N solution react with dioxygen to afford almost exclusively the simple two-electron oxidation product 3,5-di-tert-butylbenzoquinone (DBQ), which is discerned from the appearance and increase in intensity of the electronic spectral band around 400 nm, and smaller amounts of cleavage products. Interestingly, in DMF/piperidine the amount of quinone product decreases and those of the cleavage products increase illustrating that the stronger base piperidine enhances the concentration of the catecholate adduct. The rates of both dioxygenation and quinone formation observed in DMF/Et(3)N solution vary in the order 1 > 3 > 4 < 2 suggesting that the ligand steric hindrance to molecular oxygen attack, the Lewis acidity of the iron(iii) center and the ability of the complexes to rearrange the Fe-O phenolate bonds to accommodate the catecholate substrate dictate the extent of interaction of the complexes with substrate and hence determine the rates of reactions. This is in line with the observation of DBSQ/H(2)DBC reduction wave for the adduct [Fe(L2)(DBC)](-) at a potential (E(1/2): -0.285 V) more positive than those for the adducts of 1, 3 and 4 (E(1/2): -0.522 to -0.645 V).

The first crystallographic structures of a catechol 1,2-dioxygenase from a Gram-positive bacterium Rhodococcus opacus 1CP (Rho 1,2-CTD), a Fe(III) ion containing enzyme specialized in the aerobic biodegradation of catechols, and its adducts with catechol, 3-methylcatechol, 4-methylcatechol, pyrogallol (benzene-1,2,3-triol), 3-chlorocatechol, 4-chlorocatechol, 3,5-dichlorocatechol, 4,5-dichlorocatechol and protocatechuate (3,4-dihydroxybenzoate) have been determined and analyzed. This study represents the first extensive characterization of catechols adducts of 1,2-CTDs. The structural analyses reveal the diverse modes of binding to the active metal iron ion of the tested catechols thus allowing to identify the residues selectively involved in recognition of the diverse substrates by this class of enzymes. The comparison is further extended to the structural and functional characteristics of the other 1,2-CTDs isolated from Gram-positive and Gram-negative bacteria. Moreover the high structural homology of the present enzyme with the 3-chlorocatechol 1,2-dioxygenase from the same bacterium are discussed in terms of their different substrate specificity. The catalytic rates for Rho 1,2-CTD conversion of the tested compounds are also compared with the calculated energies of the highest occupied molecular orbital (E(HOMO)) of the substrates. A quantitative relationship (R=0.966) between the ln k(cat) and the calculated electronic parameter E(HOMO) was obtained for catechol, 3-methylcatechol, 4-methylcatechol, pyrogallol, 3-chlorocatechol, 4-chlorocatechol. This indicates that for these substrates the rate-limiting step of the reaction cycle is dependent on their nucleophilic reactivity. The discrepancies observed in the quantitative relationship for 3,5-dichlorocatechol, 4,5-dichlorocatechol and protocatechuate are ascribed to the sterical hindrances leading to the distorted binding of such catechols observed in the corresponding structures.

Catechol 1,2-dioxygenases and chlorocatechol dioxygenases are Fe(III)-dependent enzymes that do not require a reductant to perform the ortho cleavage of the aromatic ring. The reaction mechanism is common to the two enzymes, and active-site residues must play a key role in the fine-tuning of specificity. Protein engineering was applied for the first time to the catalytic pocket of a catechol 1,2-dioxygenase by site-specific and site-saturation mutagenesis with the purpose of redesigning the pocket shape for improved catalysis on bulky derivatives. Mutants were analysed for changes in kinetic parameters: variants for residue 69 show an inversion of specificity with a preference towards 4-chlorocatechol (decrease of K(M) by a factor of 20) and activity on the rarely recognised substrate 4,5-dichlorocatechol, thus creating a novel, engineered chlorocatechol dioxygenase. A L69A substitution conveys gain-of-function activity towards 4-tert-butylcatechol. Mutations of position 72 enhance k(cat) towards chlorinated substrates. The biphasic Arrhenius plot observed in A72S suggests the involvement of a dynamic switch in the fine regulation of the enzyme.

Dioxygen binding process of nonheme iron(III) center in intradiol catechol dioxygenase was investigated with CASSCF/CASPT2 method to incorporate multiconfigurational character participating in Fe-O(2) interaction. In this process, two alternative mechanisms were proposed: one is called "oxygen activation" and the other is called "substrate activation". Our CASSCF/CASPT2-calculated results support the oxygen activation. Potential energy curves and electronic structure evaluated with SA(state-averaged)-CASSCF/CASPT2 method indicate that the charge transfer directly occurs from the catecholate moiety to the dioxygen moiety in the O(2) binding process, to produce eta(1)-end-on type iron(III)-superoxo complex. This is the key step of the dioxygen activation. Interestingly, the iron center always keeps high spin d(5) character during the O(2) binding process, indicating the iron(III) center does not receive charge transfer from the catecholate moiety. However, this does not mean that the iron(III) center is not necessary to the dioxygen activation. The important role which the iron(III) center plays in catechol dioxygenase is to adjust the energy level of O(2) to induce the charge transfer from the catecholate moiety to the dioxygen moiety. Besides the eta(1)-end-on iron(III)-superoxo complex, eta(2)-side-on type iron(III)-superoxo complex is also optimized. This species is more stable than the eta(1)-end-on type iron(III)-superoxo complex, suggesting that this is considered as a stable isomer in the early stage of the catalytic cycle.

The degradation of phenol (2-30 mM) by free cells and by alginate-immobilized cells of Aureobasidium pullulans FE13 isolated from stainless steel effluents was studied in batch cultures with saline solution not supplemented with nutrients or yeast extract. The rate at which the immobilized cells degrade phenol was similar to the rate at which the suspended cells could degrade phenol, for a concentration of up to 16 mM of phenol. The maximum phenol volumetric degradation rate for 16 mM phenol was found to be 18.35 mg l(-1)h(-1) in the assays with free cells and 20.45 mg l(-1)h(-1) in the assays with alginate-immobilized cells, 18 mM phenol and cellular concentration of 0.176 g/l. At concentrations higher than this, an inhibitory effect was observed, resulting in the lowering of the phenol degradation rates. The immobilization was detrimental to the catechol 1,2-dioxygenase activity. However, the immobilized cells remained viable for a longer period, increasing the efficiency of phenol degradation. The yeast showed catechol 1,2-dioxygenase activity only after growth in the phenol, which was induced at phenol concentrations as low as 0.05 mM and up to 25 mM at 45 h of incubation at 30 degrees C. Phenol concentrations higher than 6mM were inhibitory to the enzyme. Addition of glucose, lactate, succinate, and benzoate reduced the rate at which phenol is consumed by cells. Our results suggest that inoculants based on immobilized cells of A. pullulans FE13 has potential application in the biodegradation of phenol and possibly in the degradation of other related aromatic compounds.

Objectives: Candida tropicalis yeast is a microorganism that possesses high tolerance for phenol and shows strong phenol degrading activity. This yeast is capable of utilizing phenol as the sole carbon and energy source. While the enzyme participating on the first step of phenol biodegradation, NADPH-dependent phenol hydroxylase, has already been characterized, information on the enzyme participating in the second step of its degradation, catechol 1,2-dioxygenase, is scarce. The development of the procedure suitable for catechol 1,2-dioxygenase isolation and partial characterization of this enzyme are the aims of this study., Methods: Combination of chromatography on DEAE-Sepharose and gel-permeation chromatography on Sephadex G-100 was used for isolation of cytosolic catechol 1,2-dioxygenase from C. tropicalis yeast. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel chromatography on Sephadex G-100 were used to evaluate the molecular mass of the enzyme. The enzyme activity was followed by HPLC (catechol consumption and/or cis,cis-muconic acid formation)., Results: Using the isolation procedure consisting of chromatography and re-chromatography on a column of DEAE-Sepharose and gel filtration on Sephadex G-100, catechol 1,2-dioxygenase was purified from C. tropicalis cytosol to homogeneity. Catechol 1,2-dioxygenase was found to be a homodimer with a subunit molecular mass of 30000 +/- 5000. The enzyme oxidized catechol producing cis,cis-muconic acid. The optimal temperature and pH were 30 degrees C and 7.7, respectively., Conclusions: The data are the first report showing the isolation of eukaryotic catechol 1,2-dioxygenase from C. tropicalis to homogeneity and its partial characterization.

The degradation pathways of benzoate at high concentration in Pseudomonas putida P8 were directly elucidated through mass spectrometric identification of some key catabolic enzymes. Proteins from P. putida P8 grown on benzoate or succinate were separated using two-dimensional gel electrophoresis. For cells grown on benzoate, eight distinct proteins, which were absent in the reference gel patterns from succinate-grown cells, were found. All the eight proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as catabolic enzymes involved in benzoate degradation. Among them, CatB (EC5.5.1.1), PcaI (EC2.8.3.6), and PcaF (EC2.3.1.174) were the enzymes involved in the ortho-cleavage pathway; DmpC (EC1.2.1.32), DmpD (EC3.1.1.-), DmpE (EC4.2.1.80), DmpF (EC1.2.1.10), and DmpG (EC4.1.3.-) were the meta-cleavage pathway enzymes. In addition, enzyme activity assays showed that the activities of both catechol 1,2-dioxygenase (C12D; EC1.13.11.1) and catechol 2,3-dioxygenase (C23D; EC1.13.11.2) were detected in benzoate-grown P. putida cells, undoubtedly suggesting the simultaneous expression of both the ortho- and the meta-cleavage pathways in P. putida P8 during the biodegradation of benzoate at high concentration.

A series of iron(III) complexes of the type [Fe(L)Cl3], where L is the variously N-alkyl-substituted bis(pyrid-2-ylmethyl)amine ligand such as bis(pyrid-2-ylmethyl)amine (L1), N,N-bis(pyrid-2-ylmethyl)methylamine (L2), N,N-bis(pyrid-2-ylmethyl)-n-propylamine (L3), N,N-bis(pyrid-2-ylmethyl)-iso-butylamine (L4), N,N-bis(pyrid-2-ylmethyl)-iso-propylamine (L5), N,N-bis(pyrid-2-ylmethyl)cyclohexylamine (L6), and N,N-bis(pyrid-2-ylmethyl)-tert-butylamine (L7), have been isolated and characterized by elemental analysis and spectral and electrochemical methods. The crystal structures of the complexes [Fe(L2)Cl3] 2, [Fe(L3)Cl3] 3, and the complex-substrate adduct [Fe(L5)(TCC)(NO3)] 5a, where TCC2- is the tetrachlorocatecholate dianion, have been determined by single-crystal X-ray crystallography. The complexes [Fe(L2)Cl3] 2 and [Fe(L3)Cl3] 3 possess a distorted octahedral geometry, in which the linear tridentate 3N ligands are cis-facially coordinated to the iron(III) center, and three chloride ions occupy the remaining coordination sites. The replacement of the N-methyl group in 2 by N-n-propyl group as in 3 leads to the formation of the Fe-Npy bonds and also the Fe-Cl bonds located trans to them of different lengths. The catecholate adduct 5a also possesses a distorted octahedral geometry, in which the ligand is cis-facially coordinated to iron(III) center, TCC2- is asymmetrically chelated trans to the two pyridyl moieties of the ligand, and one of the oxygen atoms of the nitrate ion occupies the sixth coordination site. All of the present complexes have been interacted with simple and substituted catechols. The catecholate adducts [Fe(L)(DBC)Cl] and [Fe(L)(DBC)(Sol)]+, where H2DBC is 3,5-di-tert-butylcatechol and Sol=H2O/CH3CN, have been generated in situ, and their spectral and redox properties and dioxygenase activities have been studied in dimethylformamide and dichloromethane solutions. All of the complexes catalyze the cleavage of H2DBC using molecular oxygen to afford both intra- and extradiol cleavage products. The formation of extradiol cleavage products is facilitated by cis-facial coordination of the 3N ligands and availability of vacant coordination site on iron(III) center for dioxygen binding. It is remarkable that the nature of the N-alkyl substituent in 3N ligands controls the regioselectivity of cleavage, with the n-propyl, iso-butyl, iso-propyl, and cyclohexyl groups enhancing the yield of extradiol products (46-68%) in dichloromethane. The rate of oxygenation depends upon the solvent and the Lewis acidity of iron(III) center as modified by the sterically demanding N-alkyl groups-length and degree of substitution. The plot of log (kO2) versus energy of the low-energy DBC2--to-iron(III) LMCT band is linear, demonstrating the importance of the Lewis acidity of the iron(III) center in dictating the rate of the dioxygenase reaction.

The structural and spectroscopic characterization of mononuclear iron(III)-catecholato complexes of ligand L4 (methyl bis(1-methylimidazol-2-yl)(2-hydroxyphenyl)methyl ether, HL4) are described, which closely mimic the enzyme-substrate complex of the intradiol-cleaving catechol dioxygenases. The tridentate, tripodal monoanionic ligand framework of L4 incorporates one phenolato and two imidazole donor groups and thus well reproduces the His2Tyr endogenous donor set. In fact, regarding the structural features of [FeIII(L4)(tcc)(H2O)] (5.H2O, tcc = tetrachlorocatechol) in the solid state, the complex constitutes the closest structural model reported to date. The iron(III)-catecholato complexes mimic both the structural features of the active site and its spectroscopic characteristics. As part of its spectroscopic characterization, the electron paramagnetic resonance (EPR) spectra were successfully simulated using a simple model that accounts for D strain. The simulation procedure showed that the observed g = 4.3 line is an intrinsic part of the EPR envelope of the studied complexes and should not necessarily be attributed to a highly rhombic impurity. [FeIII(L4)(dtbc)(H2O)] (dtbc = 3,5-di-tert-butylcatechol) was studied with respect to its dioxygen reactivity, and oxidative cleavage of the substrate was observed. Intradiol- and extradiol-type cleavage products were found in roughly equal amounts. This shows that an accurate structural model of the first-coordination sphere of the active site is not sufficient for obtaining regioselectivity.

Catechol 1,2-dioxygenase gene, calA, from naphthalene-degrading plasmid pND6-1 of Pseudomonas putida ND6, was cloned and expressed in Escherichia coli. Enzymic properties of the expressed product were investigated. The results indicated that the Km and Vmax of the enzyme are 0.019 mol/L and 1.434 mol/(min x mg), respectively. The enzyme possessed a thermal stability and 93.7% activity was retained after incubating at 50 degrees C for 45 min. Fe2+ could enhance the enzyme activity by 292%. The enzyme displayed a lower activity against 4-chlorocatechol and belongs to group I of catechol 1,2-dioxygenases. When naphthalene was used as a substrate for growth of strain ND6, catechol 1, 2-dioxygenase and catechol 2,3-dioxygenase activities were both detected in their crude extract. However, when strain ND6 was grown on benzoate, rho-hydroxybenzoic acid or phenylacetic acid as a sole source of carbon the activity of catechol 1,2-dioxygenase was much higher than that of catechol 2,3-dioxygenase. These indicated that strain ND6 is able to metabolize naphthalene by catechol meta- and ortho-cleavage pathways. When benzoate, rho-hydroxybenzoic acid and phenylacetic acid were used as growth substrates, strain ND6 degrades these compounds only by catechol ortho-cleavage pathway.

The crystal structure of the 3-chlorocatechol 1,2-dioxygenase from the Gram-positive bacterium Rhodococcus opacus (erythropolis) 1CP, a Fe(III) ion-containing enzyme specialized in the aerobic biodegradation of 3-chloro- and methyl-substituted catechols, has been solved by molecular replacement techniques using the coordinates of 4-chlorocatechol 1,2-dioxygenase from the same organism (PDB code 1S9A) as a starting model and refined at 1.9 A resolution (R(free) 21.9%; R-factor 17.4%). The analysis of the structure and of the kinetic parameters for a series of different substrates, and the comparison with the corresponding data for the 4-chlorocatechol 1,2-dioxygenase isolated from the same bacterial strain, provides evidence of which active site residues are responsible for the observed differences in substrate specificity. Among the amino acid residues expected to interact with substrates, only three are altered Val53(Ala53), Tyr78(Phe78) and Ala221(Cys224) (3-chlorocatechol 1,2-dioxygenase(4-chlorocatechol 1,2-dioxygenase)), clearly identifying the substitutions influencing substrate selectivity in these enzymes. The crystallographic asymmetric unit contains eight subunits (corresponding to four dimers) that show heterogeneity in the conformation of a co-crystallized molecule bound to the catalytic non-heme iron(III) ion resembling a benzohydroxamate moiety, probably a result of the breakdown of recently discovered siderophores synthesized by Gram-positive bacteria. Several different modes of binding benzohydroxamate into the active site induce distinct conformations of the interacting protein ligands Tyr167 and Arg188, illustrating the plasticity of the active site origin of the more promiscuous substrate preferences of the present enzyme.

The detection of aromatic compounds from pesticides and industrial wastewater has become of great interest, since these compounds withstand chemical oxidation and biological degradation, accumulating in the environment. In this work, a highly sensitive biosensor for detecting catechol was obtained with the immobilization of Cl-catechol 1,2-dioxygenase (CCD) in nanostructured films. CCD layers were alternated with poly(amidoamine) generation 4 (PAMAM G4) dendrimer using the electrostatic layer-by-layer (LbL) technique. Circular dichroism (CD) measurements indicated that the immobilized CCD preserved the same conformation as in solution. The thickness of the very first CCD layers in the LbL films was estimated at ca. 3.6 nm, as revealed by surface plasmon resonance (SPR). PAMAM/CCD 10-bilayer films were employed in detecting diluted catechol solutions using either an optical or electrical approach. Due to the mild immobilization conditions employed, especially regarding the pH and ionic strength of the dipping solutions, CCD remained active in the films for periods longer than 3 weeks. The optical detection comprised absorption experiments in which the formation of cis-cis muconic acid, resulting from the reaction between CCD and catechol, was monitored by measuring the absorbance at 260 nm after film immersion in catechol solutions. The electrical detection was carried out using LbL films deposited onto gold-interdigitated electrodes immersed in aqueous solutions at different catechol concentrations. Using impedance spectroscopy in a broad frequency range (1Hz-1kHz), we could detect catechol in solutions at concentrations as low as 10(-10) M.

Catechol 1,2-dioxygenase was first studied by Hayaishi and colleagues in 1950. In 1967, catechol 1,2-dioxygenase from Pseudomonas arvilla C-1 (PaCTD) was chosen as a model system for the catecholic intradiol dioxygenases due to its activity, stability and expression level. Here we report the 2.65 A structure of the betabeta isozyme of PaCTD. The structure supports the hypothesis first made by Vetting and Ohlendorf [The 1.8A crystal structure of catechol 1,2-dioxygenase reveals a novel hydrophobic helical zipper as a subunit linker, Struct. Fold. Des. 8 (2000) 429-440.] that the catechol 1,2-dioxygenases are lipid binding proteins. The 5 amino-terminal helices involved in dimerization and forming the lipid binding site are shown to be plastic in their positions and orientations. The sequence differences between the alpha and beta polypeptides are located at the part of the monomers distant from dimerization surface and thus permit the formation of the 3 isozymes (alphaalpha, alphabeta, and betabeta) of PaCTD. The reported inactivation by sulfhydryl-modifying reagents is explained by the structure. The 10-residue Helix F (residues 203-212) is proposed to be central in communicating between the lipid binding site and the active site.

In this brief review, I recollect my experiences of how the studies of pyrocatechase and salicylate hydroxylase led to the isolation and revelation of P450cam. Those experiences were instrumental in the separation, purification, and characterization of the two forms of adrenal cortex mitochondrial P450.

The basis of the respective regiospecificities of intradiol and extradiol dioxygenase is poorly understood and may be linked to the protonation state of the bidentate-bound catechol in the enzyme/substrate complex. Previous ultraviolet resonance Raman (UVRR) and UV-visible (UV-vis) difference spectroscopic studies demonstrated that, in extradiol dioxygenases, the catechol is bound to the Fe(II) as a monoanion. In this study, we use the same approaches to demonstrate that, in catechol 1,2-dioxygenase (C12O), an intradiol enzyme, the catechol binds to the Fe(III) as a dianion. Specifically, features at 290 nm and 1550 cm(-1) in the UV-vis and UVRR difference spectra, respectively, are assigned to dianionic catechol based on spectra of the model compound, ferric tris(catecholate). The UVRR spectroscopic band assignments are corroborated by density functional theory (DFT) calculations. In addition, negative features at 240 nm in UV-vis difference spectra and at 1600, 1210, and 1175 cm(-1) in UVRR difference spectra match those of a tyrosinate model compound, consistent with protonation of the axial tyrosinate ligand when it is displaced from the ferric ion coordination sphere upon substrate binding. The DFT calculations ascribe the asymmetry of the bound dianionic substrate to the trans donor effect of an equatorially ligated tyrosinate ligand. In addition, the computations suggest that trans donation from the tyrosinate ligand may facilitate charge transfer from the substrate to yield the iron-bound semiquinone transition state, which is capable of reacting with dioxygen. In illustrating the importance of ligand trans effects in a biological system, the current study demonstrates the power of combining difference UVRR and optical spectroscopies to probe metal ligation in solution.

In recent work, it was shown that V-containing polyoxometalates such as (n-Bu4N)7SiW9V3O40 or (n-Bu4N)9P2W15V3O62, as well as eight other V-containing precatalysts tested, evolve to a high activity, long catalytic lifetime (> or = 30,000-100,000 total turnovers) 3,5-di-tert-butylcatechol dioxygenase, in which Pierpont's complex [VO(DBSQ)(DTBC)]2 (where DBSQ is 3,5-di-tert-butylsemiquinone and DTBC is the 3,5-di-tert-butylcatecholate dianion) was identified as a common catalyst or catalyst resting state (Yin, C.-X.; Finke, R. G. Vanadium-Based, Extended Catalytic Lifetime Catechol Dioxygenases: Evidence For a Common Catalyst. J. Am. Chem. Soc. 2005, 127 (25), 9003-9013). Herein, those findings are followed up by studies aimed at answering the following questions about this record catalytic lifetime 3,5-di-tert-butylcatechol dioxygenase catalyst: (i) What is the key to how V leaches from, for example, seemingly robust V-containing polyoxometalate precatalysts? (ii) What is the key to the sigmoidal, apparently autocatalytic kinetics observed? (iii) What can be learned about the underlying reactions that form [VO(DBSQ)(DTBC)]2? (iv) Finally, do the answers to (i-iii) lead to any broader insights or concepts? Key findings from the present work include the fact that the reaction involves a novel, autoxidation-product-induced dioxygenase, that is, one in which the undesired autoxidation of the 3,5-di-tert-butylcatechol substrate to the corresponding benzoquinone and H2O2 turns on the desired dioxygenase catalysis via a V-leaching process which eventually yields Pierpont's complex, [VO(DBSQ)(DTBC)]2. Plausible reactions en route to [VO(DBSQ)(DTBC)]2 consistent with the kinetic data, the role of H2O2, and the relevant literature are provided. The results provide a prototype example of the little observed but likely more general concept of an autoxidation-product-initiated reaction. The results also provide considerable simplification of, and insight into, the previously disparate literature of V-based 3,5-di-tert-butylcatechol dioxygenase catalysis.