Function of different amino acid residues in the reaction mechanism of gentisate 1,2-dioxygenases deduced from the analysis of mutants of the salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans.

The genome of the α-proteobacterium Pseudaminobacter salicylatoxidans codes for a ferrous iron containing ring-fission dioxygenase which catalyzes the 1,2-cleavage of (substituted) salicylate(s), gentisate (2,5-dihydroxybenzoate), and 1-hydroxy-2-naphthoate. Sequence alignments suggested that the "salicylate 1,2-dioxygenase" (SDO) from this strain is homologous to gentisate 1,2-dioxygenases found in bacteria, archaea and fungi. In the present study the catalytic mechanism of the SDO and gentisate 1,2-dioxygenases in general was analyzed based on sequence alignments, mutational and previously performed crystallographic studies and mechanistic comparisons with "extradiol- dioxygenases" which cleave aromatic nuclei in the 2,3-position. Different highly conserved amino acid residues that were supposed to take part in binding and activation of the organic substrates were modified in the SDO by site-specific mutagenesis and the enzyme variants subsequently analyzed for the conversion of salicylate, gentisate and 1-hydroxy-2-naphthoate. The analysis of enzyme variants which carried exchanges in the positions Arg83, Trp104, Gly106, Gln108, Arg127, His162 and Asp174 demonstrated that Arg83 and Arg127 were indispensable for enzymatic activity. In contrast, residual activities were found for variants carrying mutations in the residues Trp104, Gly106, Gln108, His162, and Asp174 and some of these mutants still could oxidize gentisate, but lost the ability to convert salicylate. The results were used to suggest a general reaction mechanism for gentisate-1,2-dioxygenases and to assign to certain amino acid residues in the active site specific functions in the cleavage of (substituted) salicylate(s).

Biodegradation of naphthalenesulphonate polymers: the potential of a combined application of fungi and bacteria.

The potential of several fungi and their synergy with bacterial biomasses were evaluated as a solution for the removal of 2-naphthalensulphonic acid polymers (2-NSAPs) from petrochemical wastewater, characterized by a chemical oxygen demand (COD) greater than 9000 mg/L. The ability of fungi to grow on 2-NSAP mixtures was preliminarily investigated using a solid medium, and then the action of the selected strains, both in suspended and immobilized form, was evaluated in terms of degradation, depolymerization, sorption and an increase in biodegradability of 2-NSAP. Among the 25 fungi evaluated two, in particular, Bjerkandera adusta and Pleurotus ostreatus, have been found to significantly depolymerize 2-NSAP yielding to the corresponding monomer (2-naphthalenesulphonic acid, 2-NSA), which has been further degraded by a bacterial consortia selected in a wastewater treatment plant (WWTP). The fungal treatment alone was able to reduce the COD value up to 44%, while activated sludge removed only 9% of the initial COD. In addition, the combined treatment (fungi and bacteria) allowed an increase in the COD removal up to 62%.

The salicylate 1,2-dioxygenase as a model for a conventional gentisate 1,2-dioxygenase: crystal structures of the G106A mutant and its adducts with gentisate and salicylate.

UNLABELLED: The salicylate 1,2-dioxygenase (SDO) from the bacterium Pseudaminobacter salicylatoxidans BN12 is a versatile gentisate 1,2-dioxygenase (GDO) that converts both gentisate (2,5-dihydroxybenzoate) and various monohydroxylated substrates. Several variants of this enzyme were rationally designed based on the previously determined enzyme structure and sequence differences between the SDO and the 'conventional' GDO from Corynebacterium glutamicum. This was undertaken in order to define the structural elements that give the SDO its unique ability to dioxygenolytically cleave (substituted) salicylates. SDO variants M103L, G106A, G111A, R113G, S147R and F159Y were constructed and it was found that G106A oxidized only gentisate; 1-hydroxy-2-naphthoate and salicylate were not converted. This indicated that this enzyme variant behaves like previously known 'conventional' GDOs. Crystals of the G106A SDO variant and its complexes with salicylate and gentisate were obtained under anaerobic conditions, and the structures were solved and analyzed. The amino acid residue Gly106 is located inside the SDO active site cavity but does not directly interact with the substrates. Crystal structures of G106A SDO complexes with gentisate and salicylate showed a different binding mode for salicylate when compared with the wild-type enzyme. Thus, salicylate coordinated in the G106A variant with the catalytically active Fe(II) ion in an unusual and unproductive manner because of the inability of salicylate to displace a hydrogen bond that was formed between Trp104 and Asp174 in the G106A variant. It is proposed that this type of unproductive substrate binding might generally limit the substrate spectrum of 'conventional' GDOs. DATABASE: Structural data are available in the Protein Data Bank databases under the accession numbers 3NST, 3NWA, 3NVC.

X-ray structures of 4-chlorocatechol 1,2-dioxygenase adducts with substituted catechols: new perspectives in the molecular basis of intradiol ring cleaving dioxygenases specificity.

The crystallographic structures of 4-chlorocatechol 1,2-dioxygenase (4-CCD) complexes with 3,5-dichlorocatechol, protocatechuate (3,4-dihydroxybenzoate), hydroxyquinol (benzen-1,2,4-triol) and pyrogallol (benzen-1,2,3-triol), which act as substrates or inhibitors of the enzyme, have been determined and analyzed. 4-CCD from the Gram-positive bacterium Rhodococcus opacus 1CP is a Fe(III) ion containing enzyme specialized in the aerobic biodegradation of chlorocatechols. The structures of the 4-CCD complexes show that the catechols bind the catalytic iron ion in a bidentate mode displacing Tyr169 and the benzoate ion (found in the native enzyme structure) from the metal coordination sphere, as found in other adducts of intradiol dioxygenases with substrates. The analysis of the present structures allowed to identify the residues selectively involved in recognition of the diverse substrates. Furthermore the structural comparison with the corresponding complexes of catechol 1,2-dioxygenase from the same Rhodococcus strain (Rho-1,2-CTD) highlights significant differences in the binding of the tested catechols to the active site of the enzyme, particularly in the orientation of the aromatic ring substituents. As an example the 3-substituted catechols are bound with the substituent oriented towards the external part of the 4-CCD active site cavity, whereas in the Rho-1,2-CTD complexes the 3-substituents were placed in the internal position. The present crystallographic study shed light on the mechanism that allows substrate recognition inside this class of high specific enzymes involved in the biodegradation of recalcitrant pollutants.

The generation of a 1-hydroxy-2-naphthoate 1,2-dioxygenase by single point mutations of salicylate 1,2-dioxygenase--rational design of mutants and the crystal structures of the A85H and W104Y variants.

Key amino acid residues of the salicylate 1,2-dioxygenase (SDO), an iron (II) class III ring cleaving dioxygenase from Pseudaminobacter salicylatoxidans BN12, were selected, based on amino acid sequence alignments and structural analysis of the enzyme, and modified by site-directed mutagenesis to obtain variant forms with altered catalytic properties. SDO shares with 1-hydroxy-2-naphthoate dioxygenase (1H2NDO) its unique ability to oxidatively cleave monohydroxylated aromatic compounds. Nevertheless SDO is more versatile with respect to 1H2NDO and other known gentisate dioxygenases (GDOs) because it cleaves not only gentisate and 1-hydroxy-2-naphthoate (1H2NC) but also salicylate and substituted salicylates. Several enzyme variants of SDO were rationally designed to simulate 1H2NDO. The basic kinetic parameters for the SDO mutants L38Q, M46V, A85H and W104Y were determined. The enzyme variants L38Q, M46V, A85H demonstrated higher catalytic efficiencies toward 1-hydroxy-2-naphthoate (1H2NC) compared to gentisate. Remarkably, the enzyme variant A85H effectively cleaved 1H2NC but did not oxidize gentisate at all. The W104Y SDO mutant exhibited reduced reaction rates for all substrates tested. The crystal structures of the A85H and W104Y variants were solved and analyzed. The substitution of Ala85 with a histidine residue caused significant changes in the orientation of the loop containing this residue which is involved in the active site closing upon substrate binding. In SDO A85H this specific loop shifts away from the active site and thus opens the cavity favoring the binding of bulkier substrates. Since this loop also interacts with the N-terminal residues of the vicinal subunit, the structure and packing of the holoenzyme might be also affected.

Crystallization and preliminary X-ray crystallographic analysis of hydroquinone dioxygenase from Sphingomonas sp. TTNP3.

Hydroquinone dioxygenase (HQDO), a novel Fe(II) ring-fission dioxygenase from Sphingomonas sp. strain TTNP3 which oxidizes a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes, has been crystallized. The enzyme is an α(2)ß(2) heterotetramer constituted of two subunits of 19 and 38 kDa. Diffraction-quality crystals of HQDO were obtained using the sitting-drop vapour-diffusion method at 277 K from a solution consisting of 16% PEG 4000, 0.3 M MgCl(2), 0.1 M Tris pH 8.5. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 88.4, b = 125.4, c = 90.8 Å, ß = 105.3°. The asymmetric unit contained two heterotetramers, i.e. four copies of each of the two different subunits related by noncrystallographic 222 symmetry. A complete data set extending to a maximum resolution of 2.5 Šwas collected at 100 K using a wavelength of 0.980 Å.

Reaction intermediates and redox state changes in a blue laccase from Steccherinum ochraceum observed by crystallographic high/low X-ray dose experiments.

The crystal structure of a blue laccase from Steccherinum ochraceum has been solved at 2.0Å of resolution using a classic data acquisition from a single crystal. The overall structural features are typical of this class of enzymes, however, distances inside the trinuclear copper cluster are indicative of a reduction of the metal centers induced by free electrons produced during the X-ray data collection. UV-visible spectra collected during the X-ray exposure support the progressive reduction of the metal centers. In order to better detect the reduction progression steps in the trinuclear copper site, a multicrystal data collection strategy based on a systematic spread of the X-ray dose over many crystals has been employed. This approach is based on collecting multicrystal data sets, then combining the slices of the individual data sets experiencing the same radiation dose to obtain composite complete data sets at progressively higher doses. Applying this technique, we have been able to capture sequential frames of the enzyme during the metal centers and molecular oxygen reduction mechanism obtaining a three-dimensional movie of the X-ray-driven catalytic conversion of the molecular oxygen in the active site of laccase: first, the copper ions reduction, then the molecular oxygen binding and its reductive splitting, thus allowing to reconstruct the entire catalytic cycle for multicopper oxidases.

Enzymatic decolorization of spent textile dyeing baths composed by mixtures of synthetic dyes and additives.

The effects of different components of real dyeing bath formulations, such as the equalizing and fixing additives-acids, salts, and surfactants-on the decolorization catalyzed by Funalia trogii enzymatic extracts, were investigated to understand their influence on the recalcitrance to biodegradation of this type of wastewater. The decolorization of selected dyes and dye mixtures after tissue dyeing was performed in the presence/absence of auxiliary compounds. All spent dyeing baths were enzymatically decolorized to different extents, by the addition of extracts containing laccase only or laccase plus cellobiose dehydrogenase. Whereas surfactant auxiliaries, in some instances, inhibit the decolorization of spent dyeing baths, in several occurrences the acid/salt additives favor the enzymatic process. In general, the complete spent dyeing formulations are better degraded than those containing the dyes only. The comparison of extracellular extracts obtained from spent straws from the commercial growth of Pleurotus sp. mushrooms with those from F. trogii reveals similar decolorization extents thus allowing to further reduce the costs of bioremediation.

Crystal structures of salicylate 1,2-dioxygenase-substrates adducts: A step towards the comprehension of the structural basis for substrate selection in class III ring cleaving dioxygenases.

The crystallographic structures of the adducts of salicylate 1,2-dioxygenase (SDO) with substrates salicylate, gentisate and 1-hydroxy-2-naphthoate, obtained under anaerobic conditions, have been solved and analyzed. This ring fission dioxygenase from the naphthalenesulfonate-degrading bacterium Pseudaminobacter salicylatoxidans BN12, is a homo-tetrameric class III ring-cleaving dioxygenase containing a catalytic Fe(II) ion coordinated by three histidine residues. SDO is markedly different from the known gentisate 1,2-dioxygenases or 1-hydroxy-2-naphthoate dioxygenases, belonging to the same class, because of its unique ability to oxidatively cleave salicylate, gentisate and 1-hydroxy-2-naphthoate. The crystal structures of the anaerobic complexes of the SDO reveal the mode of binding of the substrates into the active site and unveil the residues which are important for the correct positioning of the substrate molecules. Upon binding of the substrates the active site of SDO undergoes a series of conformational changes: in particular Arg127, His162, and Arg83 move to make hydrogen bond interactions with the carboxyl group of the substrate molecules. Unpredicted concerted displacements upon substrate binding are observed for the loops composed of residues 40-43, 75-85, and 192-198 where several aminoacidic residues, such as Leu42, Arg79, Arg83, and Asp194, contribute to the closing of the active site together with the amino-terminal tail (residues 2-15). Differences in substrate specificity are controlled by several residues located in the upper part of the substrate binding cavity like Met46, Ala85, Trp104, and Phe189, although we cannot exclude that the kinetic differences observed could also be generated by concerted conformational changes resulting from amino-acid mutations far from the active site.

Differential decolorization of textile dyes in mixtures and the joint effect of laccase and cellobiose dehydrogenase activities present in extracellular extracts from Funalia trogii.

The largest part of the bio-decolorization investigations have been performed to date on a single dye without exploring the behavior in complex mixtures as the real dyeing baths. Therefore, mixtures of dyes belonging to azo and anthraquinonic classes, chosen among the most utilized in textile wool dyeing, were employed for comparative enzymatic decolorization studies using the extracellular extracts from the white rot fungus Funalia trogii, to understand how the concomitant presence of more than one dye could influence their degradation course and yield. Fungal extracts containing laccase activity only were capable to partially decolorize dyes mixtures from the different classes analyzed. The deconvolution of the decolorization with time allowed to monitor the degradation of the single dyes in the mixtures evidencing a time dependent differential decolorization not observed for the singles alone. Some dyes in the blend were in fact decolorized only when the most easily converted dyes were largely transformed. These experiments would allow to help the dyeing factories in the selection of the most readily degraded dyes. Since F. trogii grown on different media and activators shows diverse levels of expression of the redox enzymes laccase and cellobiose dehydrogenase (CDH), the dyes mixtures recalcitrant to decolorization by laccase activity alone, were subjected to the combined action of extracts containing laccase and CDH. The use of CDH, in support to the activity of laccase, resulted in substantial decolorization increases (>84%) for all the refractory dyes mixtures.

Catechol 1,2-dioxygenase from the Gram-positive Rhodococcus opacus 1CP: quantitative structure/activity relationship and the crystal structures of native enzyme and catechols adducts.

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.

Role of surfactants in optimizing fluorene assimilation and intermediate formation by Rhodococcus rhodochrous VKM B-2469.

Biodegradation of fluorene by Rhodococcus rhodochrous VKM B-2469 was investigated and optimized by adding non-ionic surfactants to the liquid media. The utilization of 1-1.5% Tween 60 or 1% Triton X100 allowed to solubilize 1 mM fluorene over 150 times more than in water medium (from 9-11 microM to above 1.5 mM at 28 degrees C). We observed that Tween 60 was useful to enhance the fluorene biodegradation rates further supporting R. rhodochrous VKM B-2469 growth as an additional carbon source and to decrease fluorene toxicity for bacterial cells whereas Triton X100 resulted to be toxic for this strain. An additional enzyme induction step before starting the bioconversion process and the increase of incubation temperature during fluorene bioconversion led to further improvements in rates of fluorene utilization and formation of its intermediates. In the optimized conditions 1 mM fluorene was degraded completely within 24h of incubation. Some intermediates in fluorene degradation built up during the process reaching maxima of 31% for 9-hydroxyfluorene, 2.1% for 9-fluorenone and 1.9% for 2-hydroxy-9-fluorenone (starting from 1 mM substrate). In the presence of Tween 60 the appearance and following conversion of 2-hydroxy-9-fluorenone was observed for R. rhodochrous VKM B-2469 revealing the existence of a new pathway of 9-fluorenone bioconversion.

Salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans: crystal structure of a peculiar ring-cleaving dioxygenase.

The crystallographic structure of salicylate 1,2-dioxygenase (SDO), a new ring fission dioxygenase from the naphthalenesulfonate-degrading strain Pseudaminobacter salicylatoxidans BN12, which oxidizes salicylate to 2-oxohepta-3,5-dienedioic acid by a novel ring fission mechanism, has been solved by molecular replacement techniques and refined at 2.9 A resolution (R(free) 26.1%; R-factor 19.3%). SDO is a homo-tetramer member of type III extradiol-type dioxygenases with a subunit topology characteristic of the bicupin beta-barrel folds. The catalytic center contains a mononuclear iron(II) ion coordinated to three histidine residues (His119, His121, and His160), located within the N-terminal domain in a solvent-accessible pocket. SDO is markedly different from the known gentisate 1,2-dioxygenases (GDO) or 1-hydroxy-2-naphthoate dioxygenase because of its unique ability to oxidatively cleave numerous salicylates, gentisates and 1-hydroxy-2-naphthoate with high catalytic efficiency. The comparison of the structure and substrate specificity for a series of different substrates with the corresponding data for several GDOs and the docking of salicylates/gentisates in the active site of SDO, allowed the identification of several active site residues responsible for differences of substrate specificity. In particular, a more defined electron density of the N-terminal region allowed the discovery of a novel structure fragment in SDO previously unobserved in GDO. This region contributes several residues to the active site that influence substrate specificity for both of these enzymes. Implications on the catalytic mechanism are discussed.

Combined action of a bacterial monooxygenase and a fungal laccase for the biodegradation of mono- and poly-aromatic hydrocarbons.

The combined action of a wide substrate range toluene o-xylene monooxygenase from Pseudomonas sp. OX1, able to convert many aromatic compounds into mono- and di-hydroxylated derivatives, and fungal laccases from Pleurotus ostreatus which oxidize these hydroxylated products yielding polymers with reduced toxicity is described. This strategy permits to overcome many of the substrate specificity problems and dead end toxic products formation generally encountered in complex bacterial biodegradation pathways. Toluene and naphthalene degradations were tested as representative of mono- and poly-aromatic pollutants. The combined biological action was optimized in micellar and microemulsion systems able to increase the bioavailability of the hydrophobic aromatic pollutants. This approach allows efficient hydroxylations of hydrophobic substrates thus favoring the further action of fungal oxidases.

Fungal laccase, cellobiose dehydrogenase, and chemical mediators: combined actions for the decolorization of different classes of textile dyes.

Dyes belonging to the mono-, di-, tri- and poly-azo as well as anthraquinonic and mono-azo Cr-complexed classes, chosen among the most utilized in textile applications, were employed for a comparative enzymatic decolorization study using the extracellular crude culture extracts from the white rot fungus Funalia (Trametes) trogii grown on different culture media and activators able to trigger different levels of expression of oxidizing enzymes: laccase and cellobiose dehydrogenase. Laccase containing extracts were capable to decolorize some dyes from all the different classes analyzed, whereas the recalcitrant dyes were subjected to the combined action of laccase and the chemical mediator HBT, or laccase plus cellobiose dehydrogenase. Correlations among the decolorization degree of the various dyes and their electronic and structural diversities were rationalized and discussed. The utilization of cellobiose dehydrogenase in support to the activity of laccase for the decolorization of azo textile dyes resulted in substantial increases in decolorization for all the refractory dyes proving to be a valid alternative to more expensive and less environmentally friendly chemical treatments of textile dyes wastes.

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

BACKGROUND: 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 O2 reduction to H2O. RESULTS: The structure of a laccase from the white-rot fungus Lentinus (Panus) tigrinus, a glycoenzyme involved in lignin biodegradation, was solved at 1.5 A. 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, micro-eta 1:eta 1 peroxide 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 (micro3-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. CONCLUSION: 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.

Crystal structure of 3-chlorocatechol 1,2-dioxygenase key enzyme of a new modified ortho-pathway from the Gram-positive Rhodococcus opacus 1CP grown on 2-chlorophenol.

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.

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.

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

The blue laccase from the white-rot basidiomycete fungus Panus tigrinus, an enzyme involved in lignin biodegradation, has been crystallized. P. tigrinus laccase crystals grew within one week at 296 K using the sitting-drop vapour-diffusion method in 22%(w/v) PEG 4000, 0.2 M CaCl2, 100 mM Tris-HCl pH 7.5. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 54.2, b = 111.6, c = 97.1, beta = 97.7 degrees , and contain 46% solvent. A complete native data set was collected to 1.4 A resolution at the copper edge. Molecular replacement using the Coprinus cinereus laccase structure (PDB code 1hfu) as a starting model was performed and initial electron-density maps revealed the presence of a full complement of copper ions. Model refinement is in progress. The P. tigrinus laccase structural model exhibits the highest resolution available to date and will assist in further elucidation of the catalytic mechanism and electron-transfer processes for this class of enzymes.

Structural roles of the active site iron(III) ions in catechol 1,2-dioxygenases and differential secondary structure changes in isoenzymes A and B from Acinetobacter radioresistens S13.

The reversible active site metal ion removal process for two catechol 1,2-dioxygenase isoenzymes (IsoA and IsoB) isolated from Acinetobacter radioresistens S13 has been monitored using circular dichroism and fluorescence spectroscopic techniques. IsoA and IsoB are homodimers, containing one iron(III) ion per subunit. Their amino acid sequence identity is 48.4%. Previous experiments suggested that structural diversities could be responsible for the differential thermal and pH stabilities of the two isoenzymes and of their distinct demetallation kinetics. The far-UV CD spectra of IsoA and IsoB catechol 1,2-dioxygenases from A. radioresistens S13 provide information on their secondary structures. IsoB appears to have a content of alpha-helices higher than IsoA. Upon metal ion removal, both proteins reversibly lose part of their secondary structure following distinct pathways. CD spectra simulations allowed us to estimate the content of alpha-helices, beta-sheets, and turns for each isoenzyme and to monitor the secondary structure rearrangements. The metal ion withdrawal has large influence on the secondary structure: in particular a significant reduction of alpha-helices content is observed for both isoenzymes. Intrinsic fluorescence emission spectra clearly support such results, adding information on the local environment changes of the tryptophan residues. The positioning of Trp250 in IsoB has been shown to be of particular interest for monitoring the local structure changes occurring upon metal ion removal. For the first time these studies allow to underline the role of active site iron ions on dioxygenases folding and stability, further evidencing the differences in structural assembling between the two isoenzymes from A. radioresistens S13.