Crystal Structure of 4-Chlorocatechol 1,2-Dioxygenase from the Chlorophenol-utilizing Gram-positive Rhodococcus opacus 1CP

The crystal structure of the 4-chlorocatechol 1,2-dioxygenase from the Gram-positive bacterium Rhodococcus opacus (erythropolis) 1CP, a Fe(III) ion-containing enzyme involved in the aerobic biodegradation of chloroaromatic compounds, has been solved by multiple wavelength anomalous dispersion using the weak anomalous signal of the two catalytic irons (1 Fe/257 amino acids) and refined at a 2.5 A resolution (R(free) 28.7%; R factor 21.4%). The analysis of the structure and its comparison with the structure of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus ADP1 (Ac 1,2-CTD) highlight significant differences between these enzymes. The general topology of the present enzyme comprises two catalytic domains (one for each subunit) related by a noncrystallographic 2-fold axis and separated by a common alpha-helical zipper motif consisting of five N-terminal helices from each subunit; furthermore the C-terminal tail is shortened significantly with respect to the known Ac 1,2-CTD. The presence of two phospholipids binding in a hydrophobic tunnel along the dimer axis is shown here to be a common feature for this class of enzyme. The active site cavity presents several dissimilarities with respect to the known catechol-cleaving enzyme. The catalytic nonheme iron(III) ion is bound to the side chains of Tyr-134, Tyr-169, His-194, and His-196, and a cocrystallized benzoate ion, bound to the metal center, reveals details on a novel mode of binding of bidentate inhibitors and a distinctive hydrogen bond network with the surrounding ligands. Among the amino acid residues expected to interact with substrates, several are different from the corresponding analogs of Ac 1,2-CTD: Asp-52, Ala-53, Gly-76, Phe-78, and Cys-224; in addition, regions of largely conserved amino acid residues in the catalytic cleft show different shapes resulting from several substantial backbone and side chain shifts. The present structure is the first of intradiol dioxygenases that specifically catalyze the cleavage of chlorocatechols, key intermediates in the aerobic catabolism of toxic chloroaromatics

Crystal structure of a blue laccase from Lentinus tigrinus: evidences for intermediates in the molecular oxygen reductive splitting by multicopper oxidases

<p>Abstract</p> <p>Background</p> <p>Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in pathogenesis, immunogenesis and morphogenesis of organisms and in the metabolic turnover of complex organic substances. They catalyze the coupling between the four one-electron oxidations of a broad range of substrates with the four-electron reduction of dioxygen to water. These catalytic processes are made possible by the contemporaneous presence of at least four copper ion sites, classified according to their spectroscopic properties: one type 1 (T1) site where the electrons from the reducing substrates are accepted, one type 2 (T2), and a coupled binuclear type 3 pair (T3) which are assembled in a T2/T3 trinuclear cluster where the electrons are transferred to perform the O₂reduction to H₂O.</p> <p>Results</p> <p>The structure of a laccase from the white-rot fungus <it>Lentinus (Panus) tigrinus</it>, a glycoenzyme involved in lignin biodegradation, was solved at 1.5 Å. It reveals a asymmetric unit containing two laccase molecules (A and B). The progressive reduction of the copper ions centers obtained by the long-term exposure of the crystals to the high-intensity X-ray synchrotron beam radiation under aerobic conditions and high pH allowed us to detect two sequential intermediates in the molecular oxygen reduction pathway: the "peroxide" and the "native" intermediates, previously hypothesized through spectroscopic, kinetic and molecular mechanics studies. Specifically the electron-density maps revealed the presence of an end-on bridging, μ-η₁:η₁peroxide ion between the two T3 coppers in molecule B, result of a two-electrons reduction, whereas in molecule A an oxo ion bridging the three coppers of the T2/T3 cluster (μ3-oxo bridge) together with an hydroxide ion externally bridging the two T3 copper ions, products of the four-electrons reduction of molecular oxygen, were best modelled.</p> <p>Conclusion</p> <p>This is the first structure of a multicopper oxidase which allowed the detection of two intermediates in the molecular oxygen reduction and splitting. The observed features allow to positively substantiate an accurate mechanism of dioxygen reduction catalyzed by multicopper oxidases providing general insights into the reductive cleavage of the O-O bonds, a leading problem in many areas of biology.</p

Crystal Structure of the Hydroxyquinol 1,2-Dioxygenase from Nocardioides simplex 3E, a Key Enzyme Involved in Polychlorinated Aromatics Biodegradation

Hydroxyquinol 1,2-dioxygenase (1,2-HQD) catalyzes the ring cleavage of hydroxyquinol (1,2,4-trihydroxybenzene), a central intermediate in the degradation of aromatic compounds including a variety of particularly recalcitrant polychloro- and nitroaromatic pollutants. We report here the primary sequence determination and the analysis of the crystal structure of the 1,2-HQD from Nocardioides simplex 3E solved at 1.75 A resolution using the multiple wavelength anomalous dispersion of the two catalytic irons (1 Fe/293 amino acids). The catalytic Fe(III) coordination polyhedron composed by the side chains of Tyr164, Tyr197, His221, and His223 resembles that of the other known intradiol-cleaving dioxygenases, but several of the tertiary structure features are notably different. One of the most distinctive characteristics of the present structure is the extensive openings and consequent exposure to solvent of the upper part of the catalytic cavity arranged to favor the binding of hydroxyquinols but not catechols. A co-crystallized benzoate-like molecule is also found bound to the metal center forming a distinctive hydrogen bond network as observed previously also in 4-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus 1CP. This is the first structure of an intradiol dioxygenase specialized in hydroxyquinol ring cleavage to be investigated in detail

XAS characterization of the active sites of novel intradiol ring-cleaving dioxygenases: hydroxyquinol and chlorocatechol dioxygenases

AbstractThe intradiol cleaving dioxygenases hydroxyquinol 1,2-dioxygenase (HQ1,2O) from Nocardiodes simplex 3E, chlorocatechol 1,2-dioxygenase (ClC1,2O) from Rhodococcus erythropolis 1CP, and their anaerobic substrate adducts (hydroxyquinol-HQ1,2O and 4-chlorocatechol-ClC1,2O) have been characterized through X-ray absorption spectroscopy. In both enzymes the iron(III) is pentacoordinated and the distance distribution inside the Fe(III) first coordination shell is close to that already found in the extensively characterized protocatechuate 3,4-dioxygenase. The coordination number and the bond lengths are not significantly affected by the substrate binding. Therefore it is confirmed that the displacement of a protein donor upon substrate binding has to be considered a general step valid for all intradiol dioxygenases

A New Modified ortho Cleavage Pathway of 3-Chlorocatechol Degradation by Rhodococcus opacus 1CP: Genetic and Biochemical Evidence

The 4-chloro- and 2,4-dichlorophenol-degrading strain Rhodococcus opacus 1CP has previously been shown to acquire, during prolonged adaptation, the ability to mineralize 2-chlorophenol. In addition, homogeneous chlorocatechol 1,2-dioxygenase from 2-chlorophenol-grown biomass has shown relatively high activity towards 3-chlorocatechol. Based on sequences of the N terminus and tryptic peptides of this enzyme, degenerate PCR primers were now designed and used for cloning of the respective gene from genomic DNA of strain 1CP. A 9.5-kb fragment containing nine open reading frames was obtained on pROP1. Besides other genes, a gene cluster consisting of four chlorocatechol catabolic genes was identified. As judged by sequence similarity and correspondence of predicted N termini with those of purified enzymes, the open reading frames correspond to genes for a second chlorocatechol 1,2-dioxygenase (ClcA2), a second chloromuconate cycloisomerase (ClcB2), a second dienelactone hydrolase (ClcD2), and a muconolactone isomerase-related enzyme (ClcF). All enzymes of this new cluster are only distantly related to the known chlorocatechol enzymes and appear to represent new evolutionary lines of these activities. UV overlay spectra as well as high-pressure liquid chromatography analyses confirmed that 2-chloro-cis,cis-muconate is transformed by ClcB2 to 5-chloromuconolactone, which during turnover by ClcF gives cis-dienelactone as the sole product. cis-Dienelactone was further hydrolyzed by ClcD2 to maleylacetate. ClcF, despite its sequence similarity to muconolactone isomerases, no longer showed muconolactone-isomerizing activity and thus represents an enzyme dedicated to its new function as a 5-chloromuconolactone dehalogenase. Thus, during 3-chlorocatechol degradation by R. opacus 1CP, dechlorination is catalyzed by a muconolactone isomerase-related enzyme rather than by a specialized chloromuconate cycloisomerase

Crystallization and preliminary structure analysis of the blue laccase from the ligninolytic fungus Panus tigrinus

Blue laccase from the white-rot basidiomycete P. tigrinus, an enzyme involved in lignin biodegradation, has been crystallized. The crystals obtained give diffraction data at 1.4 Å, the best resolution to date for this class of enzymes, which may assist in further elucidation of the catalytic mechanism of multicopper oxidases