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

Transformation of high concentrations of chlorophenols by the white-rot basidiomycete Trametes versicolor immobilized on nylon mesh

Free-cell cultures of Trametes versicolor were compared with cultures immobilized on nylon mesh in a 2-litre bioreactor for transformation of pentachlorophenol (PCP) and 2,4-dichlorophenol (2,4-DCP), added at intervals to the liquid culture medium over a period of 816 hrs. Increasing amounts of PCP from 200 ppm to 2000 ppm added batchwise to cultures permitted acclimatization of the fungus to these toxic pollutants. A total addition of 2000 ppm of 2,4-DCP and 3400 ppm PCP were removed from the immobilized cultures with 85% of 2,4-DCP and 70% of PCP transformed by enzymes (laccase and Mn-peroxidase), 5% 2,4-DCP and 28% PCP adsorbed by the biomass and 10% 2,4-DCP and 2% PCP retained in the medium at the termination of the fermentation after 1020 hrs. In contrast free-cell cultures in the same medium with the same addition regime of PCP and 2,4-DCP, transformed 20% 2,4-DCP and 12% PCP by enzyme action, adsorbed 58% 2,4-DCP and 80% PCP by the biomass, and retained 22% 2,4-DCP and 8% PCP in the medium. The use of nylon mesh as an immobilization matrix for removal of PCP and 2,4-DCP facilitates more efficient removal of chlorophenols and can be adapted to scale-up for application of large volumes of chlorophenol-containing aqueous effluents

A 2,5-Dihydroxybenzoic Acid–Gelatin Conjugate Inhibits the Basal and Hsp90-Stimulated Migration and Invasion of Tumor Cells

The extracellular cell surface-associated and soluble heat shock protein 90 (Hsp90) is known to participate in the migration and invasion of tumor cells. Earlier, we demonstrated that plasma membrane-associated heparan sulfate proteoglycans (HSPGs) bind the extracellular Hsp90 and thereby promote the Hsp90-mediated motility of tumor cells. Here, we showed that a conjugate of 2,5-dihydroxybenzoic acid with gelatin (2,5-DHBA&ndash;gelatin), a synthetic polymer with heparin-like properties, suppressed the basal (unstimulated) migration and invasion of human glioblastoma A-172 and fibrosarcoma HT1080 cells, which was accompanied by the detachment of a fraction of Hsp90 from cell surface HSPGs. The polymeric conjugate also inhibited the migration/invasion of cells stimulated by exogenous soluble native Hsp90, which correlated with the inhibition of the attachment of soluble Hsp90 to cell surface HSPGs. The action of the 2,5-DHBA&ndash;gelatin conjugate on the motility of A-172 and HT1080 cells was similar to that of heparin. The results demonstrate a potential of the 2,5-DHBA&ndash;gelatin polymer for the development of antimetastatic drugs targeting cell motility and a possible role of extracellular Hsp90 in the suppression of the migration and invasion of tumor cells mediated by the 2,5-DHBA&ndash;gelatin conjugate and heparin

(A) and (B) Representations of the Fo-Fc difference Fourier omit map for the T2/T3 active site of molecules A and B of L respectively, each flanked by the corresponding schematic pictures

<p><b>Copyright information:</b></p><p>Taken from "Crystal structure of a blue laccase from : evidences for intermediates in the molecular oxygen reductive splitting by multicopper oxidases"</p><p>http://www.biomedcentral.com/1472-6807/7/60</p><p>BMC Structural Biology 2007;7():60-60.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2064922.</p><p></p> The electron density is contoured at 2.2 σ. (C) Schematic representation of the catalytic mechanism of multicopper oxidases including the intermediates observed in the present structural study and previous spectroscopic, kinetic, and structural investigations

Alignment of the L blue laccase amino acid sequence with other laccase sequences

<p><b>Copyright information:</b></p><p>Taken from "Crystal structure of a blue laccase from : evidences for intermediates in the molecular oxygen reductive splitting by multicopper oxidases"</p><p>http://www.biomedcentral.com/1472-6807/7/60</p><p>BMC Structural Biology 2007;7():60-60.</p><p>Published online 26 Sep 2007</p><p>PMCID:PMC2064922.</p><p></p> Lt – laccase [ and Pdb code: ]; Tve – [gi:21730581, pdb:]; Tvi – []; Pc – []; Ft – []; So – laccase [unpublished gene-xray]; Cc – [pdb:]; Rl – [pdb:]; Ts – []; Ma – []; Tov – [] ; Mt – []; Bs – Cota [pdb:]. Positions identical in all sequences are marked with a black background. Regions, in which the sequences are similar are marked with light grey