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.

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.

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.

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.

Exploring new molecular architectures for anion recognition: synthesis and ATP binding properties of new cyclam-based ditopic polyammonium receptors.

Synthesis and characterization of three new polyamine receptors, composed of a cyclam unit (cyclam=1,4,8,11-tetraazacyclotetradecane) linked by a 2,6-dimethylpyridinyl spacer to the linear polyamines 1,4,8,11-tetraazaundecane (L1py), 1,4,7-triazaheptane (L2py), and to a quaternary ammonium group (L3py(+)), are reported. All receptors form highly charged polyammonium cations at neutral pH, suitable for anion recognition studies. ATP recognition was analyzed by using potentiometric, calorimetric, (1)H and (31)P NMR measurements in aqueous solution. All receptors form 1:1 adducts with ATP in aqueous solution, stabilized by charge-charge and hydrogen-bonding interactions between their ammonium groups and the anionic triphosphate chain of ATP. The binding ability of the three receptors for ATP increases in the order of L3py(+)

Proton and metal binding by cyclen-based highly rigid cryptands.

The basicity properties of the two cryptands L1 and L2, featuring, respectively, a dibenzofuran or a diphenyl ether moiety bridging the 1,7 positions of a 1,4,7,10-tetraazacyclododecane macrocycle (cyclen) have been studied by means of potentiometric, UV-vis and fluorescence emission measurements. Both ligands show a high basicity in the first protonation step, the first basicity constant of L1 being too high to be measured in aqueous solution. The crystal structure of {[HL1]L1}(+) shows that the NH(2)(+) group is involved in an intramolecular hydrogen bonding network, which justifies the observed high basicity in solution. (1)H, (13)C NMR, UV-vis and fluorescence emission measurements show that, among first row divalent metal cations, both L1 and L2 selectively bind in acetonitrile Cu(II) and Zn(II), which are encapsulated within the ligand cavities. Zn(II) coordination is accompanied by a remarkable increase of the fluorescence emission of the ligands, pointing out that the molecular architecture displayed by L1 and L2 can be used to develop new OFF/ON chemosensors for this metal cation.

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.

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.

ATP recognition and sensing with a phenanthroline-containing polyammonium receptor.

A new polyammonium receptor is able to selectively recognise and sense ATP among triphosphate nucleotides, thanks to ATP-induced quantitative quenching of its fluorescence emission.