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Research article Open Access Crystal structure of a blue laccase from Lentinus tigrinus: evidences for intermediates in the molecular oxygen reductive splitting by multicopper oxidases Marta Ferraroni1, Nina M Myasoedova2, Vadim Schmatchenko2, Alexey A Leontievsky2, Ludmila A Golovleva2, Andrea Scozzafava1 and Fabrizio Briganti*1

Address: 1Department of Chemistry University of Florence, Via della Lastruccia 3, Sesto Fiorentino, 50019 Florence, Italy and 2G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms Russian Accademy of Science, Pushchino, Moscow Region, 1422920, Russia Email: Marta Ferraroni - [email protected]; Nina M Myasoedova - [email protected]; Vadim Schmatchenko - [email protected]; Alexey A Leontievsky - [email protected]; Ludmila A Golovleva - [email protected]; Andrea Scozzafava - [email protected]; Fabrizio Briganti* - [email protected] * Corresponding author

Published: 26 September 2007 Received: 28 March 2007 Accepted: 26 September 2007 BMC Structural Biology 2007, 7:60 doi:10.1186/1472-6807-7-60 This article is available from: http://www.biomedcentral.com/1472-6807/7/60 © 2007 Ferraroni et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract 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 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:η1 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 (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. 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.

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Background range of physiological functions such as lignolysis, lignin Laccases (benzenediol oxygen oxidoreductase; EC synthesis, morphogenesis, pathogenesis and detoxifica- 1.10.3.2) are glycosilated multicopper oxidases. Since tion. their discovery more than one century ago in the Japanese tree Rhus venicifera [1], laccases have been found to be Owing to their high and non-specific oxidation capacities, widely distributed among plants, where they are involved to the lack of a requirement for cofactors and to the use of in the wounding response and the synthesis of lignin, and readily available oxygen as an electron acceptor laccases subsequently they were discovered to be present in are useful biocatalysts with some established and lots of insects, bacteria and widely diffused in basidiomyceteous emerging biotechnological applications such as biob- and ascomyceteous fungi [2]. leaching, xenobiotics bioremediation, textile dyes decolorization, biosensors, food industry etc. [13-15]. The simplest reactions catalyzed by laccases are those in which a vast set of substrates, typically phenols and These multicopper oxidases are generally monomeric arylamino compounds, is oxidized to the corresponding glycoproteins containing about 500 amino acids arranged radical species by direct interaction with their active site in 3 β-barrel domains assembled to model three spectro- [3] and accompanied by the reduction of molecular oxy- scopically distinct copper binding catalytic sites: one type gen to water. Due to their very broad substrate range they 1 (T1, blue copper, characterized by a strong absorption at are implicated in a extensive series of functions such as 600 nm), one type 2 (T2, normal copper) and one type 3 pathogenesis, immunogenesis and morphogenesis of (T3, EPR-silent antiferromagnetically coupled dinuclear organisms and in the metabolic turnover of complex coppers)[16,17]. Four one-electron oxidations of the organic substances such as lignin, humic matter, and toxic reducing substrates mentioned above which occur at the xenobiotics [4]. T1 site on the protein surface are coupled to the four-electrons reduction of dioxygen to water which occurs at the For instance, white-rot fungi degrade wood lignin using a internal T2/T3 cluster [18,19]: combination of specialized intra- and extra-cellular - + enzymes [5]. Lignin, the most common polymer on earth O2 + 4 e + 4 H → 2 H2O which provides the structural component of the plant cell wall, is a heterogeneous biopolymer composed by phenyl The unique kinetic and spectral features of copper/dioxy- propanoid units linked by various non-hydrolyzable C-C- gen intermediates in multicopper oxidases have been and C-O- bonds [6]. The ligninolytic system of white-rot investigated into details mainly by Reinhammar and Solo- fungi was thought to be mainly composed by lignin-per- mon groups [18,20]. These studies led to several hypoth- oxidase and manganese-peroxidase [7]. Laccases, incapa- eses of molecular mechanisms for the four-electrons ble of directly cleaving the non-phenolic bonds of lignin, reduction of dioxygen to water by this class of enzymes. were not considered as significant components of the ligninolytic system, despite the secretion of large quanti- X-ray structural studies on ascorbate oxidase, ceruloplas- ties of laccases by the vast majority of white-rot fungi min and on several fungal and bacterial laccases have fur- under ligninolytic conditions. More recently it was discov- ther allowed the rationalization of important structural ered that white-rot fungi laccases can enlarge their sub- and functional aspects of multicopper oxidases such as strate range and are then able to oxidize compounds with the positioning of the T1 site, the electron transfer path- a redox potential exceeding their own, such as non-phe- ways between T1 and the T2/T3 cluster coppers, the oxy- nolic benzylalcohols [8-10]. In fact their catalytic compe- gen and water channels, and some of the substrates/ tences can be further extended to substrates which are not inhibitor interactions [16,19,21-28]. oxidized directly, either because they are too large to pen- etrate into the enzyme active site or because they have a Regarding fungal laccases at first, the extensive micro- redox potential higher than the laccase itself. Nature over- heterogeneity, presumably caused by variable glycosyla- comes these limitations with the utilization of mediators, tion of these enzymes, hindered successful crystallization which are suitable compounds that act as intermediate but deglycosylation performed to obtain high quality dif- substrates for the laccase: the oxidized radical forms of fracting crystals resulted in the loss of copper as in the case which are sufficiently stable to leave the enzyme site and of Ducros et al. which reported the crystal structure of a react with the bulky or high redox-potential substrate tar- laccase from the fungus Coprinus cinereus at 1.68 Å resolu- gets. This finding led to the discovery that laccase-media- tion[16] in a form devoid of the type 2 copper and there- tor systems effectively play a major role in the fore in a catalytically incompetent state. More recently the biodegradation of lignin and recalcitrant aromatic pollut- x-ray structures of laccases from the fungi Trametes versi- ants [10-12]. These non enzymatic routes of oxidative color (1.9 Å resolution), Melanocarpus albomyces (2.4 Å res- polymerizing or depolymerizing reactions are vital in a olution), Rigidoporus lignosus (1.7 Å resolution) Cerrena

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maxima (1.9 Å resolution), and the spore-coat laccase reverse primer based on internal conservative C-terminal from Bacillus subtilis (1.7 Å resolution) have been amino acid sequence of basidiomycete laccases. Therefore described [21-24,29]. the deduced amino acid sequence of L. tigrinus blue laccase is missing C-terminal residues. Furthermore the elec- Substrate and dioxygen binding was investigated for the tron density maps allowed to correct the identity of a few Bacillus subtilis endospore-coat laccase (substrate: ABTS) other residues: in position 16: V is observed instead of S, and for the Trametes versicolor laccase (substrate: 2,5-xylid- in position 164: K is present instead of L, and in position ine) [30,31]. 459: D occurs instead of E.

Nevertheless several focal points still need to be eluci- Overall structure dated from the structural standpoint including the nature The crystal structure of LtL was solved at 1.5 Å resolution of the intermediates in the oxygen reduction pathway. by using the molecular replacement technique and employing, as a starting model, the coordinates of the In particular the biotechnological application of laccases, Coprinus cinereus laccase (pdb code: 1HFU). The schematic aiming at the development of various industrial oxidative structure and folding topology of LtL, a monomeric glob- processes going from the production of fine chemicals ular glycoprotein with overall dimensions 70 × 60 × 50 Å, and pharmaceuticals to bioremediation of contaminated is shown in Figure 2A. It exhibits a molecular architecture soil and water, would receive great impulse to improve- organized in three sequentially arranged cupredoxin-like ments from the complete comprehension of the catalytic domains; each of them has a greek key β-barrel topology, mechanism of multicopper oxidases, and in particular of strictly related to the small copper proteins like azurin and their redox potential and substrate selectivity control and plastocyanin and common to all the members of the blue a detailed characterization of the high resolution molecu- multicopper oxidase family, like the ascorbate oxidase lar structure of such enzymes will surely help in achieving and the mammalian ceruloplasmin. Domain 1 includes such aims. residues 1–141, domain 2 residues 142–303, and domain 3 residues 304–498. As observed in Figure 1LtL exhibits We present here the structural analysis at the highest reso- relevant structural similarities with several basidiomycete- lution available to date of an active laccase from Lentinus ous fungal laccases and particularly with the enzyme from tigrinus (hereafter LtL) a white rot lignin decomposing Trametes versicolor [23] (78% of sequence identity), the fungus able to biodegrade high concentrations of mix- superposition of the Cα main chain atoms of the two tures of chlorophenols such as 2,4-dichlorophenol, 2,4,6- structures gives an rmsd of 0.515 Å. On the contrary major TCP, and pentachlorophenol [32,33]. Furthermore the differences are noticed in the comparison with laccases particular conditions utilized for x-ray diffraction data col- from plants, bacteria and even with ascomyceteous fungi lection have allowed the detection of the structure of two (see Figure 1). sequential intermediates in the molecular oxygen reduction pathway: the "peroxide" and the "native" intermedi- Of the six putative N-glycosylation sites (consensus ates the latter only hypothesized through spectroscopic, sequence N-X-T/S) only at Asn54, Asn376, and Asn435 kinetic and molecular mechanics calculations studies. the density maps indicate the presence of glycosilaytion. The corresponding electronic densities were modelled to Results and discussion two di(n-acetyl-D-glucosamine) and five branched α-D- Primary sequence mannose moieties bound to Asn54, one n-acetyl-D-glu- The nucleotide sequence of L. tigrinus laccase gene was cosamine bound to Asn376 (but detected only in mole- obtained as reported in the experimental procedures, the cule A) and one di(n-acetyl-D-glucosamine) moiety deduced sequence resulted to be composed by 463 amino bound to Asn435. acids (accession code: AAX07469). Since the electron density maps are of very good quality the polypeptide chain The structure is further stabilized by two sulphur bridges, has been traced throughout the molecule. This allowed us one between Cys85 and Cys487 from domains 1 and 3 to obtain the entire sequence from the x-ray data, this respectively and the other between Cys117 and Cys 205 resulting in a 498 amino acids chain (see Figure 1). The from domains 1 and 2 correspondingly. comparison of the sequence derived from the gene and that obtained from the x-ray data reveals that in the DNA Substrate binding site sequence the residues corresponding to the c-terminal end The structure of LtL does not exhibit any peculiar feature and exactly from positions 464 to 498 were missing: at the T1 active site of the enzyme where the reducing sub- VVMAEDIPNTVNANPVPQAWSNLCPTYDALEPSNE. The strate binds to transfer electrons to the T1 copper, located sequence of L. tigrinus blue laccase gene lacks some 3' end in domain 3. The binding pocket for reducing substrates nucleotides as in the gene amplifying we have used the of LtL is a relatively large superficial zone near the T1 cop-

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10 20 30 40 50 60 70 80 90 100 110 120 ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| Lt ------A VG P ------V AD L T V T N AN I V PD G F - E R A A I V V N N V F P A P L I T Lt Tve ------G I G P ------V AD L T I T N A A V S PD G F - S RQ A V V V N G G T PG P L I T Tve Tvi M S R F H S L L A F V V A S L T A V AH AG I G P ------V AD L T I T N A A V S PD G F - S RQ A V V V N G G T PG P L I T Tvi Pc M S R F Q S L F F F V L V S L T A V AN A A I G P ------V AD L T L T N AQ V S PD G F - A R E A V V V N G I T P A P L I T Pc Ft M A R F Q S L L T F I T L S L V A S V Y A A I G P ------V AD L T I S N G A V S PD G F - S RQ A I L V N D V F P S P L I T Ft So ------VQ I G P ------V T D L H I VN AD I V PD G F - V R P A VN A G G T F PG P V I A So Cc ------Q I VN S ------VD T M T L T N AN V S PD G F - T R AG I L V N G VH - G P L I R Cc Rl M P S F A S L K S L V V L S L T S L S - L A A T V ------A L D L H I L N AN L D PD G T G A R S A V T A E G T T I A P L I T Rl Ts - - M L S S I T L L P L L A A V S T P A F A A V R ------N Y K F D I KN VN V A PD G F - Q R P I V S V N G L V PG T L I T Ts Ma M K T F T S A L A L V VG M L A PG A V V A A P P S T P AQ RD L V E L R E A R Q E G G KD L R P R E P T CN T P S N R A CWS D G F D I N T D Y E V S T PD T G V T Q S Y V F N L T E VD N WMG PD G V V K E K VM L I N G N I MG PN I V Ma Tov ------VD VH N ------Y T F V L Q E KN F T KW C S - T K S M L V V N G S F PG P T I T Tov Mt M K S F I S A A T L L VG I L T P - S V A A A P P S T P E Q RD L L V P I T E R E E - - A A V K A R Q Q S CN T P S N R A CWT D G YD I N T D Y E VD S PD T G V V R P Y T L T L T E VD N WT G PD G V V K E K VM L V N N S I I G P T I F Mt Bs M T L E K F VD A L P I PD T L K P VQ Q S K E K ------T Y Y E V T M E E C T H Q L H RD L P - P T R L WG Y N G L F PG P T I E Bs

130 140 150 160 170 180 190 200 210 220 230 240 ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| Lt G N MG D N F Q L N L VN Q M T N H T M L ------K T T S I H WH G F F Q KG T N WAD G P A F I N Q C P I A S G N S F L - - - YD F Q V PG Q AG T F W YH S H L S T Q - - - - Y CD G L RG P F V V YD PN D PH Lt Tve G N MG D R F Q L N V I D N L T N H T M L ------K S T S I H WH G F F Q KG T N WAD G P A F I N Q C P I S S G H S F L - - - YD F Q V PD Q AG T F W YH S H L S T Q - - - - Y CD G L RG P F V V YD PN D P A Tve Tvi G N MG D R F Q L N V I D N L T N H T M V ------K S T S I H WH G F F Q KG T N WAD G P A F I N Q C P I S S G H S F L - - - YD F Q V PD Q AG T F W YH S H L S T Q - - - - Y CD G L RG P F V V YD PN D P A Tvi Pc G N KG D R F Q L N V I D Q L T N H T M L ------K T S S I H WH G F F Q Q G T N WAD G P A F VN Q C P I A S G H S F L - - - YD F Q V PD Q AG T F W YH S H L S T Q - - - - Y CD G L RG P F V V YD PN D PH Pc Ft G N KG D R F Q L N V I D N M T N H T M L ------K S T S I H WH G F F Q H G T N WAD G P A F VN Q C P I S T G H A F L - - - YD F Q V PD Q AG T F W YH S H L S T Q - - - - Y CD G L RG P I V V YD PQ D PH Ft So G N VG D N F Q I V T F N Q L I E C S M L ------VH T S I H WH G E F Q KG T N WAD G P A F I T Q C P I I VG N S F S - - - YN F N V PG H AG T YW YH S H L T T Q - - - - Y CD G L RG P F V V YD PN D PD So Cc G G KN D N F E L N V VN D L D N P T M L ------R P T S I H WH G L F Q RG T N WAD G AD G VN Q C P I S PG H A F L - - - Y K F T P AG H AG T F W YH S H F G T Q - - - - Y CD G L RG P M V I YD D N D PH Cc Rl G N I D D R F Q I N V I D Q L T D AN M R ------R A T S I H WH G F F Q AG T T E MD G P A F VN Q C P I I PN E S F V - - - YD F V V PG Q AG T YW YH S H L S T Q - - - - Y CD G L RG A F V V YD PN D PH Rl Ts AN KG D T L R I N V T N Q L T D P S M R ------R A T T I H WH G L F Q A T T AD E D G P A F V T Q C P I AQ N L S Y T - - - Y E I P L H G Q T G T MW YH AH L A S Q - - - - Y VD G L RG P L V I YD PN D PH Ts Ma AN WG D T V E V T V I N N L V T N ------G T S I H WH G I H Q KD T N L H D G AN G V T E C P I P P K G G Q R - - - T Y R WR A RQ YG T S W YH S H F S AQ - - - - YG N G V VG T I Q I N G - - - P A Ma Tov A R KG D T I F VN V I N Q G K YG ------L T I H WH G V KQ P RN PWS D G P E Y I T Q C P I K PG T N F I - - - Y E V I L S T E E G T L W WH AH S D WT - - - - R A T - VH G A L V I L P AN - G T Tov Mt AD WG D T I Q V T V I N N L E T N ------G T S I H WH G L H Q KG T N L H D G AN G I T E C P I P P K G G R K - - - V Y R F K AQ Q YG T S W YH S H F S AQ - - - - YG N G V VG A I Q I N G - - - P A Mt Bs V K RN E N V Y V K WMN N L P S T H F L P I D H T I H H S D S Q H E E P E V K T V VH L H G G V T PD D S D G Y P E A WF S KD F E Q T G P Y F K R E V YH Y PN Q Q RG A I L W YH D H AM A L T R L N V Y AG L VG A Y I I H D P K E K R Bs

250 260 270 280 290 300 310 320 330 340 350 360 ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| Lt AN L YD VD D E S T V I T L AD WYH V A A K - L G P R ------F P KG AD S T L I N G L G R S - T S T P T AD L A V I S V T K G K R Y R F R L V S L S CD PN Y T F S I D S - - H Q L T V I E AD G V S T Q P V T VD S I Q I F A Lt Tve AD L YD VD N D D T V I T L VD WYH V A A K - L G P A ------F P L G AD A T L I N G KG R S - P S T T T AD L S V I S V T P G K R Y R F R L V S L S CD PN Y T F S I D G - - H N M T I I E T D S I N T A P L V VD S I Q I F A Tve Tvi AD L YD VD N D D T V I T L VD WYH V A A K - L G P A ------F P L G AD A T L I N G KG R S - P S T T T AD L S V I S V T P G K R Y R F R L V S L S CD PN Y T F S I D G - - H N M T I I E T D S I N T A P L V VD S I Q I F A Tvi Pc A S L YD I D N D D T V I T L AD WYH V A A K - L G P R ------F P F G S D S T L I N G L G R T - T G I A P S D L A V I K V T Q G K R Y R F R L V S L S CD PN H T F S I D N - - H T M T I I E AD S I N T Q P L E VD S I Q I F A Pc Ft K S L YD VD D D S T V I T L AD WYH L A A K - VG S P ------V P T - AD A T L I N G L G R S - I D T L N AD L A V I T V T K G K R Y R F R L V S L S CD PN H V F S I D G - - H S L T V I E AD S VN L K P Q T VD S I Q I F A Ft So AN L YD VD D D S T I I T L AD WYH V L A K E MG AG ------G A V T AD S T L I D G L G R T H VN S A A V P L S V I T V E V G K R Y RM R L V S I S CD PN YD F S I D G - - H D M T I I E T D G VD S Q E L T VD E I Q I F A So Cc A A L YD E D D E N T I I T L AD WYH I P A P S I Q G A ------AQ PD A T L I N G KG R Y - VG G P A A E L S I VN V E Q G K K Y RM R L I S L S CD PN WQ F S I D G - - H E L T I I E VD G E L T E P H T VD R L Q I F T Cc Rl L S L YD VD D A S T V I T I AD WYH S L S T V L F PN P N ------K A P P A PD T T L I N G L G RN S AN P S A G Q L A V V S VQ S G K R Y R F R I V S T S C F PN Y A F S I D G - - H RM T V I E VD G V S H Q P L T VD S L T I F A Rl Ts K S R YD VD D A S T V VM L E D WYH T P A P V L E KQ M F S T N N T A L L S P V PD S G L I N G KG R Y - VG G P A V P R S V I N V K R G K R Y R L R V I N A S A I G S F T F S I E G - - H R L T V I E AD G I PH Q P L P VD S F Q I Y A Ts Ma S L P YD I D L G - - V F P I T D Y Y Y R A AD D L VH F T Q N - - - - - N A P P F S D N V L I N G T A VN - PN T G E G Q Y AN V T L T P G K RH R L R I L N T S T E N H F Q V S L VN - - H T M T V I A AD M V P VN A M T VD S L F L A V Ma Tov T Y P F P P P YQ E Q T I V L A S WF K G D VM E V I T S S E E T G - - - V F P A A AD G F T I N G E L G D L YN C S K E T T Y R L S VQ P N K T Y L L R I VN A V L N E E K F F G I A K - - H T L T V V AQ D A S Y I K P I N T S Y I M I T P Tov Mt S L P YD T D L G - - V F P I S D Y Y Y S S AD E L V E L T KN - - - - - S G A P F S D N V L F N G T A KH - P E T G E G E Y AN V T L T P G R RH R L R L I N T S V E N H F Q V S L VN - - H T M T I I A AD M V P VN A M T VD S L F L G V Mt Bs L K L P S D E YD V P L L I T D R T I N E D G S L F Y P S A P E N P - - - - S P S L PN P S I V P A F C G E T I L VN G K VWP Y L E V E P - R K Y R F R V I N A S N T R T YN L S L D N G G D F I Q I G S D G G L L P R S V K L N S F S L A P Bs

370 380 390 400 410 420 430 440 450 460 470 480 ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| Lt AQ R Y S F V L N A N Q D VD N YWI R AN PN F G T T G F AD - - - - G VN S A I L R YD D AD P V E P V T N - - Q T G - T T L L L E T D - - - - - L H P L T S M P V PG N P T Q G G A - - - - - D L N L N M A F N F D G T ------Lt Tve AQ R Y S F V L E A N Q A VD N YWI R AN PN F G N VG F T G - - - - G I N S A I L R YD G A A A V E P T T T - - Q T T S T A P L N E VN - - - - - L H P L V A T A V PG S P V A G G V - - - - - D L A I N M A F N F N G T ------Tve Tvi AQ R Y S F V L E A N Q A VD N YWI R AN PN F G N VG F T G - - - - G I N S A I L R YD G A A A V E P T T T - - Q T T S T A P L N E VN - - - - - L H P L V T T A V PG S P V A G G V - - - - - D L A I N M A F N F N G T ------Tvi Pc AQ R Y S F V L D A S Q P VD N YWI R AN P A F G N T G F AG - - - - G I N S A I L R YD G A P E I E P T S V - - Q T T P T K P L N E VD - - - - - L H P L S PM P V PG S P E P G G V - - - - - D K P L N L V F N F N G T ------Pc Ft AQ R Y S F V L N A D Q D VG N YWI R A L PN S G T RN F D G - - - - G VN S A I L R YD G A A P V E P T T S - - Q T P S T N P L V E S A - - - - - L T T L E G T A A PG S P A P G G V - - - - - D L A L N M A F G F AG G ------Ft So AQ R Y S F V L N A N Q P VG N YWI R AN PN S G G E G F D G - - - - G I N S A I L R YD G A T S AD P V T V A - S T VH T K C L I E T D - - - - - L H P L S S N G V PG N PH Q G G A - - - - - D C N L N L S L G F A C G ------So Cc G Q R Y S F V L D A N Q P VD N YWI R AQ PN KG RN G L AG T F AN G VN S A I L R Y AG A AN AD P T T S - - AN PN P AQ L N E AD - - - - - L H A L I D P A A PG I P T P G A A - - - - - D V N L R F Q L G F S G G ------Cc Rl G Q R Y S V V V E A N Q A VG N YWI R AN P S N G RN G F T G - - - - G I N S A I F R YQ G A A V A E P T T S - - - Q N S G T A L N E AN - - - - - L I P L I N PG A PG N P V P G G A - - - - - D I N L N L R I G RN A T T ------A Rl Ts G Q R Y S V I V E A N Q T A AN YWI R A PM T V AG AG T N AN L D P T N V F A V L H Y E G A PN A E P T T E Q - G S A I G T A L V E E N - - - - - L H A L I N PG A PG G S A P AD V - - - - - S L N L A I G R S T VD G I ------L Ts Ma G Q R YD V V I D A S R A PD N YWF N V T - - F G G Q A A C G G S L N PH P A A I F H Y AG A PG G L P T D E G T P P VD H Q C L D T L D - - - - - V R P V V P R S V P VN S F V K R P - - - - - D N T L P V A L D L T G T ------Ma Tov G Q T MD V L F T T D Q T P S H Y YM V A S P F H D A L D T F AN - - - F S T N A I I Q YN G S Y K A P K S P F V K P L P V YN D I K A AD K F T G K L R S L A N E K F P VN V P K VN V R R I F M A V S L N I V K C AN K S CN N N I G H S T Tov Mt G Q R YD V V I E A S R T PG N YWF N V T - - F G G G L L C G G S RN P Y P A A I F H Y AG A PG G P P T D E G K A P VD H N C L D L PN - - - - - L K P V V A RD V P L S G F A K R P - - - - - D N T L D V T L D T T G T ------Mt Bs A E R YD I I I D F T A Y E G E S I I L AN S AG C G G D V N P - - - - E T D A N I MQ F R V T K P L AQ KD E S ------R K P K Y L A S Y P S VQ H E R I Q N ------Bs

490 500 510 520 530 540 550 560 570 580 590 600 ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| Lt N F F I N G E S F T P P T V P V L L Q I I S G ------AN T AQ D L L P S G S V Y S L P S N S S I E I T F P A T T - - A A PG A PH P F H L H G H V F A V V R ------S AG S T S YN YD D P V Lt Tve N F F I N G A S F T P P T V P V L L Q I I S G ------AQ N AQ D L L P S G S V Y S L P S N AD I E I S F P A T A - - A A PG A PH P F H L H G H A F A V V R ------S AG S T V YN YD N P I Tve Tvi N F F I N G T S F T P P T V P V L L Q I I S G ------AQ N AQ D L L P S G S V Y S L P S N AD I E I S F P A T A - - A A PG A PH P F H L H G H A F A V V R ------S AG S T V YN YD N P I Tvi Pc N F F I N D H T F V P P S V P V L L Q I L S G ------AQ A AQ D L V P E G S V F V L P S N S S I E I S F P A T A - - N A PG F PH P F H L H G H A F A V V R ------S AG S S V YN YD N P I Pc Ft K F T I N G A S F T P P T V P V L L Q I L S G ------AQ S AQ D L L P S G S V Y S L P AN AD I E I S L P A T A - - A A PG F PH P F H L H G H T F A V V R ------S AG S S T YN Y E N P V Ft So N F V I N G V S F T P P T V P V L L Q I C S G ------AN T G AD L L P S G S V I S L P S N S S I E I A L P AG A - - AG G - - PH P F H L H G H D F A V S E ------S A S N S T S N YD D P I So Cc R F T I N G T A Y E S P S V P T L L Q I M S G ------AQ S AN D L L P AG S V Y E L P RN Q V V E L V V P AG - - - - V L G G PH P F H L H G H A F S V V R ------S AG S S T YN F VN P V Cc Rl D F T I N G A P F I P P T V P V L L Q I L S G ------V T N PN D L L PG G A V I S L P AN Q V I E I S I PG ------G G N H P F H L H G H N F D V V R ------T PG S S V YN Y VN P V Rl Ts R F T F N N I K Y E A P S L P T L L K I L AN N ------A S N D AD F T PN E H T I V L PH N K V I G AQ H H R ------G AD H P I H L H G H V F D I V K ------S L G G - T PN Y VN P P Ts Ma P L F VWK VN G S D I N VD WG K P I I D Y I ------L T G N T S Y P V S D N I VQ VD A VD Q W T YWL I E N D P E G P F S L PH PMH L H G H D F L V L G R S PD V P A A S Q Q R F V F D P A VD L A R L N G D N P P Ma Tov S A S L N N I S F A L PQ T D V L Q A Y Y RN I S G V F G R D F P T VQ K K AN F S L N T AQ G T Q V L M I E YG E A V E I V YQ G T N - - L G A A T S H PMH L H G F N F Y L VG T G AG ------T F N N V T D P P K YN L VD P P Tov Mt P L F VWK VN G S A I N I D WG R P V VD Y V ------L T Q N T S F P PG YN I V E VN G AD Q W S YWL I E N D PG A P F T L PH PMH L H G H D F Y V L G R S PD E S P A S N E RH V F D P A RD AG L L S G AN P V Mt Bs - - - I R T L K L A G T Q D E YG R P V L L L N ------N K RWH D P V T E T P K VG T T E I W S I I N P T RG ------T H P I H L H L V S F R V L D R R P F D I A R YQ E S G E L S Y T G - - P A V P P P P S E Bs

610 620 630 640 650 660 670 680 690 700 ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| ....| .... Lt WRD V V S T G T P Q D G D N V T I R F Q T D N PG PWF L H C H I D F H L D A G F A V VM A E D - - - I PN T VN AN P V PQ AWS N L C P T YD A L E P S N E ------Lt Tve F RD V V S T G T P A AG D N V T I R F R T D N PG PWF L H C H I D F H L E A G F A V V F A E D - - - I PD V A S AN P V PQ AWS D L C P T YD A RD P S D Q ------Tve Tvi F RD V V S T G T P A AG D N V T I R F R T D N PG PWF L H C H I D F H L E A G F A V V F A E D - - - I PD V A S AN P V PQ AWS D L C P T YD A L D P S D Q ------Tvi Pc F RD V V S T G Q P - - G D N V T I R F E T N N PG PWF L H C H I D F H L D A G F A V VM A E D - - - T PD T K A AN P V PQ AWS D L C P I YD A L D P S D L ------Pc Ft Y RD V V S T G S P - - G D N V T I R F R T D N PG PWF L H C H I D F H L E A G F A V VM A E D - - - I P E V A A T N P V PQ AWS D L C P T YD A L S PD D Q ------Ft So WRD V V S I G G V - - G D N V T I R F C T D N PG PWF L H C H I D WH L D A G F A I V F A E D - - - I PN T A S AN P V P E AWS N L C P S YD S AH ------So Cc K RD V V S L G V T - - G D E V T I R F V T D N PG PWF F H C H I E F H L MN G L A I V F A E D - - - M AN T VD AN N P P V E WAQ L C E I YD D L P P E A T S I Q T V V ------Cc Rl R RD V V S I G G G - - G D N V T F R F V T D N PG PWF L H C H I D WH L E A G L A V V F A E D - - - I PN I P I AN A I S P AWD D L C P K YN AN N PD S G L A ------Rl Ts R RD V V R VG G T - - - - G V V L R F K AD N PG PWF V H C H I D C T WR L G S H L S L P R P P A R F A R V S S R S S P T M PG T S S A P S T R L F L P I C S KW ------Ts Ma R RD T T M L P AG G - - - WL L L A F R T D N PG AWL F H C H I AWH V S G G L S VD F L E R - P AD L RQ R I S Q E D E D D F N R V C D E WR A YWP T N P Y P K I D S G L K R R RWV E E S E W L V R - Ma Tov E L N T I N L P R I G - - - WA A I R F V AD N PG VWF L H C H F E RH T T E G M A T V V I V K - - - - - D G G T T N T S M L P S P A YM P P C S ------Tov Mt R RD V T M L P A F G - - - WV V L A F R AD N PG AWL F H C H I AWH V S G G L G V V Y L E R - AD D L RG A V S D AD AD D L D R L C AD WR R YWP T N P Y P K S D S G L K H R - WV E E G E W L V K A Mt Bs KG WKD T I Q AH AG E V L R I A A T F G P Y S G R Y VW H C H I L E H E D Y D MM R PMD I T D PH K ------Bs

FigureAlignment 1 of the LtL blue laccase amino acid sequence with other laccase sequences Alignment of the LtL blue laccase amino acid sequence with other laccase sequences. Lt – Lentinus (Panus) tigrinus laccase [AAX07469 and Pdb code: 2QT6]; Tve – Trametes versicolor [gi:21730581, pdb:1KYA]; Tvi – Trametes villosa [AAC41686]; Pc – Pycnoporus cinnabarinus [AAF13052]; Ft – Funalia trogii [CAC13040]; So – Steccherinum ochraceum laccase [unpublished gene- xray]; Cc – Coprinus Cinereus [pdb:1A65]; Rl – Rigidoporus Lignosus [pdb:1V10]; Ts – Thizoctonia solani [CAA91042]; Ma – Melanocarpus albomyces [CAE00180]; Tov – Toxicodendron vernicifluum [BAB63411] ; Mt – Myceliophthora thermophila [AAC93841]; Bs – Bacillus subtilis Cota [pdb:1HKZ]. Positions identical in all sequences are marked with a black background. Regions, in which the sequences are similar are marked with light grey. per depicted in Figure 2C. A number of residues mainly comparable to several known high potential laccases com- His457, the solvent exposed T1 copper ligand, and several prising that from T. versicolor [23]. surrounding amino acids are potentially involved in the interactions with the different substrates which are oxi- Two solvent channels provide access to the trinuclear cop- dized at that site; hydrophobic residues such as Phe162, per cluster which is located in the interior of the protein Phe265, Phe331, Phe 336, Phe 456, are found together structure. The first channel points towards the two T3 cop- with hydrophilic residues like Asp206, Lys164, Tyr152, per ions on one side of the T2/T3 cluster allowing to the and several prolines: Pro163, Pro207, Pro390. The variety molecular oxygen to bind to it whereas the second chan- of these residues explains the large substrate specificity nel pointing towards the T2 copper ion on the other side common to this type of enzymes and mainly related to the of the cluster permits to the water molecules produced in redox potential of the T1 copper ion (> 700 mV) which is the O2 reduction to move to the bulk solvent [22].

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Table 1: Observed coordination distances (Å) in the copper centers, copper-copper and oxygen-oxygen distances for Lentinus A B tigrinus laccase

Molecule A Molecule B

Mononuclear copper center: T1 copper

His394 ND1 2.03 His394 ND1 2.04 Cys452 SG 2.23 Cys452 SG 2.25 His457 ND1 2.01 His457 ND1 2.01 Phe462 CD2 3.68 Phe462 CD2 3.68 Ile454 CD1 3.60 Ile454 CD1 3.57

Trinuclear copper center T2/T3 C

T3(a) copper

His111 NE2 2.03 His111 NE2 1.99 His451 NE2 1.99 His451 NE2 2.02 His399 NE2 1.99 His399 NE2 2.00 OH O1 2.16 Perox O1 2.08 Oxo O2 2.91 Perox O2 2.80

T3(b) copper

D His453 NE2 2.11 His453 NE2 2.10 His66 ND1 1.99 His66 ND1 1.99 His109 NE2 2.01 His109 NE2 2.00 OH O1 2.95 Perox O1 3.08 Oxo O2 2.70 Perox O2 2.18

T2 copper

His397 NE2 1.90 His397 NE2 1.91 His64 NE2 1.92 His64 NE2 1.91 OH 2.43 OH 2.49 OH O1 4.10 Perox O1 3.06 Oxo O2 2.09 Perox O2 4.25 copperFigure(A) Schematic ions2 are representati depicted ason magenta of the structure spheres of LtL; the Copper-copper (A) Schematic representation of the structure of LtL; the copper ions are depicted as magenta spheres. (B) T2/T3 tri- T1-T3(a) 12.23 T1-T3(a) 12.24 nuclear cluster active site as observed in molecule A of LtL. T1-T3(b) 13.13 T1-T3(b) 13.14 (C) Substrate active T1 pocket residues. (D) Stereoview of T1-T2 14.85 T1-T2 14.86 the schematic representation of the four copper sites in LtL. T3(a)-T3(b) 4.94 T3(a)-T3(b) 4.91 T2-T3(a) 4.32 T2-T3(a) 4.32 T2-T3(b) 4.11 T2-T3(b) 4.09

Oxygen1-Oxygen2 Copper sites The T1 copper ion (occupancy 0.75) presents a planar tri- O1-O2 2.05 Perox O1-O2 1.43 angular coordination with two histidine (His394 and His457) and one cysteine (Cys452) residues. Two hydrophobic residues (Ile454 and Phe462) at about 3.5–3.6 Å distance (see Table 1) from the copper ion are thought to contribute to the high T1 copper redox potential observed ter through an intramolecular electron transfer pathway for this enzyme (Figures 2C and 2D). In the catalytic proc- starting from the T1 copper ligand Cys452 and subse- ess the electrons acquired by the T1 copper from the quently splitting between His451 and His453 which bind reducing substrates are then transferred to the T2/T3 clus- to the T3(a) and T3(b) copper ions respectively [34].

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Multicopper oxidases are easily reduced by addition of and acquired using synchrotron radiation, are difficult to substrates or artificial reductants but their kinetics under interpret since they most probably represent pictures of aerobic conditions are complex and difficult to interpret, the copper sites resulting from the average of the starting therefore the common strategy for the study of their cata- oxidized state and of the partially/totally reduced cop- lytic mechanism has been first the analysis of the anaero- pers/dioxygen adduct states and strictly reliant on the bic reduction steps followed then by the investigation of acquired x-ray doses and on the other experimental con- oxygen reaction stages with the reduced enzyme [20]. ditions. From the distances reported on Table 1 it can be deduced that in the present structure we mainly observe a A recently discovered alternative for the progressive reduc- fully reduced structure, the inhibition of the hydroxide/ tion of metalloproteins is the exposure of crystals to a water formation allowed to detect in molecule B an elec- high-intensity X-ray synchrotron beam. In fact, in several tronic density compatible with the presence of an end-on instances, it has been observed that synchrotron radiation bridging μ-η1:η1 peroxide moiety (O1-O2 distance 1.43 can yield the partial or total reduction of the metal ions Å) between the two T3 coppers (see Figures 3B, and 4A) depending on the particular metalloprotein studied, on [see also additional file 2]. This peroxide ion should be the data collection method and accordingly on the the result of the two-electrons reduction of dioxygen. Pre- acquired radiation dose [35,36]. This strategy, applied to vious kinetic and spectroscopic investigations have led to multicopper oxidases, could allow to observe the progres- describe the four-electrons reduced native laccase as the sive reduction of molecular oxygen and monitor the species that binds dioxygen at the T3 copper pair then gen- nature of the intermediate states. Up to date no structural erating the first intermediate in dioxygen splitting: two studies allowed to perceive the different intermediates electrons are transferred to O2 from the T3 copper centers mainly because these states are generally short living and generating a peroxide intermediate [18,20]. A ligand-field difficult to trap unless the further catalytic step is inhib- analysis also suggested that the T2 copper site becomes ited. The pH optima for LtL are ranging from 2.3 for ABTS four coordinated in the peroxy-bound form probably due (2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid), a non to the peroxide forming a bridge between the T2 and the phenolic substrate, to pH 4.5 and 5.0 for 2,6-dimethoxy- T3 copper centers. These studies headed to two possible phenol and syringaldazine respectively. Furthermore it is descriptions of the peroxide intermediate in the trinuclear well known that the activity of laccases is inhibited at high copper cluster. In one model, the peroxide internally pH values due to the formation of a strong T2 Cu-OH- bridges the T2 to the T3 copper centers with the magnetic complex which hinders the subsequent transformation of coupling between the oxidized T3 Cu centers arising from 2- + reduced oxygen to water molecules (O + 2H → H2O) antiferromagnetic coupling through one oxygen atom of [37-39]. For these reasons the present experiment was the peroxide group (Figure 4B). Alternatively, the T3 designed to be performed at high pH in order to allow the Cu(II) centers could be linked by the hydroxide bridge trapping of at least the completely reduced state of the which is also present in the resting state and the peroxide oxygen-enzyme adduct. could externally bridge the T2 Cu(I) and the T3 Cu(II) centers as hydroperoxide (Figure 4C). Only very recently a Exposing LtL crystals at pH 8.5 to high X-rays radiation computational method that combines extended X-ray doses under aerobic condition allowed us to detect two absorption fine structure (EXAFS) refinements with an intermediates in the catalyzed molecular oxygen splitting, integrated quantum mechanical and molecular mechan- most likely corresponding to the 2-electrons and 4-elec- ics method allowed to suggest that the structure with a trons reduction stages. peroxide ion in the center of the trinuclear cluster fits experimental data better than the other model [40]. The 2-electrons reduction intermediate The electron-density maps from LtL disclosed two differ- Dioxygen moieties have been observed in several previ- ent T2/T3 cluster-dioxygen arrangements in the two lac- ously collected structural data on multicopper oxidases case molecules (A and B) present into the asymmetric [24,41,42]. Since strong molecular oxygen binding is fea- unit, possibly corresponding to two different reduction sible only when copper is in the Cu(I) oxidation state states of the oxygen molecule along the reduction path- these findings indicate that the x-ray sources utilized in way. In both molecules the T2 copper center is coordi- the corresponding experiments have been able to reduce nated to His64, His397 and one OH- ligand whereas the at least the T3 copper pair. Whereas in the ABTS substrate T3 copper centers are coordinated to 3 histidine residues adduct of B. subtilis CotA laccase [31] the dioxygen is each (His111, His399, and His451 for T3(a), His66, found within the oxygen channel oriented toward one of His109, and His453 for T3(b))(See Figures 2B and 2D). the T3 copper ions, in M. albomyces laccase and B. subtilis CotA CuCl2 soaked or M502L mutant laccases, dioxygen It should be noted that differences observed among the is bound to both T3 copper ions in an nearly symmetrical structural data of multicopper oxidases published to date, side-on μ-η2:η2 binding mode (Figure 4D) [24,41,42].

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C1 C2

+O2

C3 +4H+

-2H2O +2H+ + 4 e- SLOW -H2O + 4 e-

FAST

SLOW +2H+

-H2O C5 C4

Figurerespectively,(A) and 3(B) Representations each flanked by ofth ethe corresponding Fo-Fc difference schematic Fourier pictures omit map for the T2/T3 active site of molecules A and B of LtL (A) and (B) Representations of the Fo-Fc difference Fourier omit map for the T2/T3 active site of molecules A and B of LtL respectively, each flanked by the corresponding schematic pictures. 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.

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O OH O T3 Cu(II) Cu(II) Cu(II) Cu(II) Cu(II) Cu(II) Cu(I) Cu(I) O O O O O HO T2 Cu(I) Cu(I) Cu(I) Cu(I)

H2O H2O H2O H2O

A BCD

Schematicoxide)Figure 4 representations of possible adducts of the trinuclear T2/T3 copper cluster with 2-electrons reduced dioxygen (per- Schematic representations of possible adducts of the trinuclear T2/T3 copper cluster with 2-electrons reduced dioxygen (peroxide).

Furthermore, structures of adducts with exogenous perox- gen ions it surely indicates the progress towards the ide have been obtained with ascorbate oxidase from zuc- breaking of the O-O bond and the formation of an oxo chini and CotA laccase from B. subtilis. The structure of the moiety (actually, in molecule B we observe an oxygen- CotA laccase shows the peroxide molecule asymmetrically oxygen average distance of 1.43 Å which is consistent with bound between the two T3 copper ions in a end-on bridg- a peroxide ion). Unfortunately the attempt of separating ing μ-η1:η1 mode [41] (Figure 4A), thus agreeing with the the contribution of the peroxide and oxo/hydroxyl forms observations of the present investigation for molecule B of as well as a rigorous analysis of single bond distances LtL, the only difference being that in the present structure would result in an over-interpretation of the available the peroxide ion has been generated by internal two-elec- data. trons dioxygen reduction. A similar peroxide adduct was also observed in the structure of the untreated B. subtilis The step resulting from the peroxide ion reduction to the - 2- CotA laccase M502F mutant but no possible rationaliza- H2O/OH /O level, the so-called "native" or "optical" tion was reported for such finding [42]. On the contrary intermediate, was previously investigated through spec- in ascorbate oxidase the peroxide ion was located termi- troscopic and kinetic experiments. X-ray edge, MCD spec- nally bound to only one of the T3 copper ions and ori- troscopy and magneto-structural correlations showed that ented along the oxygen channel [43]. the unusual spectra of the native intermediate are associ- ated with a fully oxidized trinuclear copper cluster with all The 4-electrons reduction intermediate three copper centers involved in strong bridging interac- In molecule A of LtL the x-ray irradiation resulted in an tions [44]. These observations conducted Solomon and electron density map that can be interpreted with the coworkers to two possible structural models of the native appearance of an oxo ion (occupancy 0.5) in the middle intermediate where the four-electrons reduction of dioxy- of the T3/T2 cluster bridging all the three coppers (μ3-oxo gen has either produced one μ3-oxo bridge or two addi- bridge with distances: O-T3(a)Cu 2.91 Å, O-T3(b)Cu 2.70 tional OH- bridges (Figures 5A and 5B respectively). More Å, and O-T2Cu 2.09 Å) together with an hydroxide ion recently Rulisek et al. suggested through quantum and (occupancy 0.75) asymmetrically bridging the two T3 molecular mechanical calculations that the μ3-oxo bridge coppers (distances: OH-T3(a)Cu 2.16 Å, and OH- should be the product of O-O bond cleavage [45,46]. T3(b)Cu 2.95 Å) possibly the products of the four-elec- Unfortunately no structural evidences were available to trons reduction of molecular oxygen (Figures 3A and 5A) confirm it. Our data shed light on the structure of the [see also additional file 2]. "native" intermediate.

In particular in molecule A the reported distances would In LtL the T3(a) and T3(b) copper ions are not symmetri- suggest that the electron density picture observed could be cally coordinated to the oxygen intermediates in both the outcome of an average between the two main interme- molecules A and B. Also in the Rigidoporus lignosus laccase diates: peroxide and hydroxyl/oxo ions. Indeed the dis- (RlL) structure [21] it has been recently observed the tance between the modeled hydroxide and oxo ions asymmetric binding of the hydroxide ion bridging the T3 resulted to be 2.05 Å; although this value does not corre- copper ions (The T3(a)-OH bond being shorter than the spond to a completely broken bond between the two oxy- T3(b)-OH bond); this was explained by assuming a

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OH OH OH T3 Cu(II) Cu(II) Cu(II) Cu(II) Cu(II) Cu(II) - - O2- OH OH T2 Cu(II) Cu(II) Cu(II)

H2O H2O H2O

SchematicFigure 5 representations of different possible adducts of the trinuclear T2/T3 copper cluster Schematic representations of different possible adducts of the trinuclear T2/T3 copper cluster. A and B: 4-electrons reduced dioxygen adducts, C: enzyme resting state.

reduced oxidation state for T2 and T3(b) copper ions, reduced in a second step, thus oxidizing the T2 and the probably caused by autoreduction phenomena induced distant T1 centers to generate the "native" intermediate by the extensive exposure to high source of X-ray radia- (Figure 3-C4) which is composed by one μ3-oxo bridge tion. This asymmetry could have a crucial role in the pro- between the T2/T3 copper centers of the trinuclear cluster gression of the dioxygen splitting. It has to be noted that and an OH- bridge between the two T3 ions (Figure 3A). the T3(a) copper results to be about 0.9 Å closer to the T1 site than the T3(b) copper. According to the theoretical Since it is known that at high pH the formation of a strong calculations performed by Kyritsis et al. [34] for ascorbate T2 Cu-OH- complex inhibits the laccase activity hindering 2- + oxidase, the particular conformation of the interposed the O + 2H → H2O reaction to occur at the trinuclear aminoacidic residues, also observed in LtL, should render copper site [37-39], working at pH 8.5 allowed the trap- the T1-T3(a) pathway up to three times more efficient ping of the oxygen reduced intermediates. than the T1-T3(b), thus explaining a differential reduction progress in the T2/T3 cluster possibly resulting in the Although the final electron density is an average of the dif- observed asymmetries. ferent oxidation states starting from the "resting" completely oxidized state to the trapped reduced oxygen In the reported investigation we have been able to discern adducts and even if the experimental conditions utilized both the peroxide and the native intermediates resulting cannot allow to precisely quantify the contributions of the from the two- and four-electrons molecular oxygen reduc- different states to the final picture it is noticeable that the tion respectively, starting from aerobic crystals in which occupancy of the oxo ion resulted to be about 0.5 suggest- the complete dioxygen turnover is inhibited by the high ing that this transient species can be definitely trapped pH due to the formation of a strong T2 Cu-OH- complex employing the above reported strategy. which hinders the subsequent transformation of reduced 2- + oxygen to water molecules (O + 2H → H2O) [37-39]. Also at low pH, the kinetic data show that both the rate of decay of the native intermediate to form the resting oxi- The structural characterization of these new intermediates dized enzyme (Figure 3-C5) and the rate of reduction of in the reduction of O2 to H2O in the multicopper oxidases the resting enzyme are too slow to be consistent with the leads now to propose a more detailed unified molecular catalytic turnover rate [37], On the contrary the "native" mechanism (Figure 3C). As in several previously reported intermediate is rapidly reduced by substrate in the cata- laccase structures the presence of the dioxygen moiety lytic reaction thus indicating that this is the fully oxidized indicates that at least the reduction of the T3 copper pair form of the enzyme which is active in catalysis [44]; only has occurred. Once reduced (Figure 3-C1) the enzyme in the absence of substrates it slowly decays to the resting reacts with molecular oxygen (Figure 3-C2) to successively state. The oxidized resting state (Figure 5C) has been usu- generate the peroxide intermediate (Figure 3-C3). Contra- ally observed in the structures of resting multicopper oxi- rily to what suggested by spectroscopic studies in the crys- dases, among them ascorbate oxidase from zucchini, and tal structures the peroxide appears to bridge only the T3 laccases from Trametes versicolor, and Bacillus subtilis CotA copper pair and not to connect the reduced T2 copper [22,23,26]. All the copper ions are apparently in an oxi- center (Figure 3B). This peroxide intermediate is further dized state.

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Conclusion 45 s and 72°C for 1 min in a GeneAmp 2400 thermocy- Laccases belong to multicopper oxidases, an important cler (PerkinElmer). PCR products were loaded on a 1% and widespread class of enzymes implicated in a extensive agarose gel, electrophoresed in Tris-acetate-EDTA buffer series of oxidative functions in pathogenesis, immuno- and a single PCR product of 2 kb was cut and purified with genesis and morphogenesis of organisms as well as in the the Wizard PCR preps purification kit (Promega). A 2 kb metabolic turnover of complex organic substances such as PCR product corresponding to the blue laccase gene with- lignin, humic matter, and toxic xenobiotics. They catalyze out the fragment encoding the signal peptide was cloned the coupling between four one-electron oxidations of a into the pBluescript II KS vector (Stratagene). Cloned spe- broad range of substrates with the four-electron reduction cific blue laccase sequences from some independent PCR of dioxygen to water. experiments were sequenced on both strands.

The structure of the blue laccase, a multicopper oxidase DNA sequencing and analysis from the white-rot basidiomycete fungus Lentinus (Panus) DNA was sequenced with the Big Dye terminator ready tigrinus, involved in lignin and xenobiotics biodegrada- reaction kit from PE-Applied Biosystems and the Applied tion, was solved at 1.5 Å. The reduction of the copper ions Biosystems DNA sequencer ABI PRISM 310 Genetic Ana- centers obtained by the long-term exposure of the crystals lyzer. Sequencing reactions were performed on Thermal to the high-intensity X-ray synchrotron beam radiation in Cycler 2700 (Applied Biosystems) according to the man- aerobic conditions and pH 8.5 allowed us to trap two ufacturer's protocols. The sequence data were processed intermediates in the molecular oxygen reduction path- and the encoded amino acid sequences were predicted way: the "peroxide" and the "native" intermediates result using the Vector NTI (InforMax, Inc.) software package. of a two- and four-electrons reduction of molecular oxy- Multiple sequence alignments of amino acid sequences gen, previously hypothesized through spectroscopic were performed by the CLUSTAL program [50]. The Gen- kinetic and molecular mechanics studies. The pictures ris- Bank accession number for the sequence of LtL is ing from the present results allow the completion of the AAX07469. structural pictures for an accurate mechanism of dioxygen reduction catalyzed by multicopper oxidases providing Protein purification general insights into the reductive cleavage of the O-O Enzyme preparations containing the blue laccase from bonds, a leading problem in many areas of biology. Lentinus tigrinus were obtained from fungal cultures grown as previously described [49]. The blue laccase was purified Methods to apparent electrophoretic homogeneity as reported and Microorganism was used for subsequent crystallization experiments. Lac- White rot basidiomycete Lentinus (Panus) tigrinus 8/18 case activity was determined quantitatively by monitoring was isolated from rotting wood in Dushanbe the oxidation of 0.2 mM ABTS (2,2_-azinobis(3-ethylben- (Tadzhikistan) and stored on malt agar slants. zthiazolinesulfonic acid) at 420 nm (extinction coefficient, 36,000 mM-1 cm-1) in the presence of 20 mM Cloning of the blue laccase gene sodium acetate, pH 5.0 at 20°C. To obtain the nucleotide sequence of blue laccase gene, the specific genomic sequence of L. tigrinus was amplified. Metal content analysis L. tigrinus genomic DNA was isolated from fungal mycelia Analysis of the protein metal content was performed by grown in Soya-glycerol media [47]. Filtered mycelium was using a PerkinElmer Optima 2000 Inductively Coupled quick-frozen and ground in liquid nitrogen and genomic Plasma AES (Atomic Emission Spectrometry) Dual Vision. DNA was purified by the procedure as described [48]. The metal content analysis was performed in triplicate on Primers for amplification of blue laccase gene sequence re-solubilized crystals of LtL; it revealed the presence of were designed based on the N terminus sequence of blue 0.7–0.8 equivalents of copper ions per metal coordina- laccase as previously reported [49] and of internal con- tion site. servative C-terminus amino acid sequence of basidiomycete laccases. The forward primer was 5'- Crystallization and data collection GCCGTCGGTCCTGTCGCCGA-3' and the reverse primers Crystals of laccase from L. tigrinus grow within one week were 5'-TGGCAGTGGAGGAACCACGGGCC-3' and 5'- at 296 K using the sitting drop vapor diffusion method. 3 GGCGAAACCAGCCTCGAGGTGGAAGTC-3'. Amplifica- μl of a 20 mg/ml protein solution were added to 2 μl of a tion of the blue laccase gene sequence from L. tigrinus solution containing 22% PEG 4000, 0.2 M CaCl2, 100 genomic DNA was performed using the forward primer mM Tris·HCl pH 8.5 [32]. Crystals belong to the mono- and one of the reverse primers and Tli DNA polymerase clinic space group P21 with unit cell dimensions a = 54.2, (Promega). PCR conditions were a first denaturing step at b = 111.6, c = 97.1, β = 97.7. Assuming two molecules per 94°C for 5 min and 30 cycles of 94°C for 45 s, 65°C for asymmetric unit the solvent content is 46% of the unit cell

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(Vm = 2.31 Å 3/Da). A complete native data set was col- Table 2: X-ray data collection and atomic model refinement lected at the BW7A beamline at the DORIS storage ring, statistics Hamburg, Germany at the copper edge (1.377 Å). 358 Space Group P21 images at 4000 KHz dose each, (for a total time of expo- Unit cell parameters sure of ~12 hours) were collected at 100 K adding 10% a (Å) 54.22 glycerol to the mother liquor as cryoprotectant at a maxi- b (Å) 111.61 mum resolution of 1.50 Å. Data processing with Denzo c (Å) 97.09 and Scalepack gave 180651 unique reflections, an overall β (°) 97.75 Wavelength (Å) 1.377 completeness of 99.1% and a Rsym of 0.062 [51]. Limiting resolution (Å) 25.3-1.5 (1.53–1.50) Unique reflections 180651 a Structure determination and refinement Rsym(%) 0.062 (0.481) Structure determination was carried out by the molecular Multiplicity 6.6 replacement technique using the laccase from C. cinereus Completeness overall (%) 99.1 (97.2) (PDB code 1hfu) as a search model (see Table 2). This 20.3 (3.3) enzyme shares 58% sequence identity with the L. tigrinus Refinement laccase. Water molecules, Cu atoms and sugars were omit- Resolution range (Å) 25.3-1.5 ted from the standard model, which was used within the Unique reflections, working/free 179673/916 program MOLREP to calculate cross-rotation and transla- Rfactor (%)b 15.0 tion function in the 10-3 Å resolution range [52]. Two Rfree(%) 18.2 clear solutions were evident, giving an R factor and corre- Non-hydrogen atoms 9602 lation coefficient of 0.469 and 0.436, respectively. The ini- Water molecules 1660 tial electron-density maps are very clear and show the r.m.s.d. bonds(Å) 0.015 presence of all four Cu atoms (see Fig. 2) and of several r.m.s.d. angles (°) 1.671 24.3 carbohydrate moieties. Values in parentheses are for the highest resolution shell. a Manual rebuilding of the model was performed using the Rsym = Σ | I - |/Σ I b program QUANTA [53]. Solvent molecules were intro- Rfactor = Σ | Fobs-Fcalc|/Σ Fobs duced automatically using ARP [54]. Refinement of the model was performed using the program Refmac 5.1.24;[52] 916 reflections (5‰ of the data) were ran- domly excluded from the refinement and used to follow the progress of the procedure with Rfree. The final refine- The final model include two independent molecules (A ment resulted in Rfactor and Rfree values of 15.0 and 18.2%, and B) related by a binary axis, 8 rameic ions, 6 calcium respectively. ions, 3 chloride ions, 2 glycerol molecules, one tartrate molecule and 1660 solvent molecules. Sulfur atoms, copper, chloride, and calcium ions were refined anisotropically. The coordination distances for the The model also include a few N-linked oligosaccharides copper ions were refined unrestrained. The occupancies of moieties at the following sites: A/B54, A376, A/B435. the copper atoms were adjusted so that their isotropic More specifically we found a di(N-acetyl-D-glucosamine) thermal parameters were refined to obtain values similar linked to residue Asn435, a N-acetyl-D-glucosamine to the local average and taking into consideration the linked to residue AsnA376 and a mannose-type glycan average content of copper ions as determined through bound to residue Asn A/B54. metal content analysis. The resulting occupancies are 0.75 for the T1 and the two T3 copper ions, and 0.5 for the T2 A double conformation was modeled for the side chains copper ion [see additional file 1]. Data processing and of residues: MetA40, SerA62, CysA117, CysA205, refinement statistics are summarized in Table 2. The over- SerA235, Gln A293, Met A310, Glu A 494, Glu A498, Met all mean B factor of the structure after refinement was B40, Cys B117, Val B231, Ser B367, Ser B375, Glu B494. 17.69 Å2 for chain A, 21.21 Å2 for chain B, 40.39 Å2 for water molecules and 24.33 Å2 for all atoms. The stereo- No evidences of crystal damage were observed after data chemistry was checked with the program PROCHECK collection completion. [55]. The 86% of the protein residues lies in the most favourite regions of the Ramachandran plot and only one Protein coordinates have been deposited with the Protein residue (Leu 57) is in a non allowed region. Data Bank (Protein Data Bank accession number 2QT6).

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Authors' contributions 9. Johannes C, Majcherczyk A: Natural mediators in the oxidation of polycyclic aromatic hydrocarbons by laccase mediator MF, AS and FB conceived of the studies, performed the systems. Appl Environ Microbiol 2000, 66:524-528. crystallographic experiments, the structural analysis and 10. Bourbonnais R, Paice MG: Oxidation of non-phenolic substrates. interpretation and the manuscript writing work, NMM, An expanded role for laccase in lignin biodegradation. FEBS Lett 1990, 267:99-102. AAL, and LAG carried out the work on enzyme purifica- 11. Mayer AM, Staples RC: Laccase: new functions for an old tion and biochemical characterization, VS performed the enzyme. Phytochemistry 2002, 60:551-565. cloning and sequencing experiments. All authors read and 12. Murugesan K: Bioremediation of paper and pulp mill effluents. Indian J Exp Biol 2003, 41:1239-1248. approved the final manuscript. 13. Couto SR, Herrera JLT: Industrial and biotechnological applications of laccases: A review. Biotechnology Advances 2006, 24:500-513. Additional material 14. Aust SD, Benson JT: The fungus among us: use of white rot fungi to biodegrade environmental pollutants. Environ Health Perspect 1993, 101:232-233. Additional file 1 15. Murugesan K, Kalaichelvan PT: Synthetic dye decolourization by Table 3 – Statistical occupation and temperature B factors for the atoms white rot fungi. Indian J Exp Biol 2003, 41:1076-1087. 16. Ducros V, Brzozowski AM, Wilson KS, Brown SH, Ostergaard P, Sch- composing the trinuclear copper cluster moiety for both molecules A and neider P, Yaver DS, Pedersen AH, Davies GJ: Crystal structure of B.The Table reports the statistical Occupation and Temperature B factors the type-2 Cu depleted laccase from Coprinus cinereus at for the atoms composing the trinuclear copper cluster moiety for both mol- 2.2 A resolution. Nat Struct Biol 1998, 5:310-316. ecules A and B. 17. Malmstrom BG: Enzymology of oxygen. Annu Rev Biochem 1982, Click here for file 51:21-59. 18. Solomon EI, Chen P, Metz M, Lee SK, Palmer AE: Oxygen Binding, [http://www.biomedcentral.com/content/supplementary/1472- Activation, and Reduction to Water by Copper Proteins. 6807-7-60-S1.pdf] Angew Chem Int Ed Engl 2001, 40:4570-4590. 19. Solomon EI, Sundaram UM, Machonkin TE: Multicopper Oxidases Additional file 2 and Oxygenases. Chem Rev 1996, 96:2563-2606. 20. Garavaglia S, Cambria MT, Miglio M, Ragusa S, Iacobazzi V, Palmieri F, Figure 6 – Representations of the 2Fo-Fc difference Fourier map with a D'Ambrosio C, Scaloni A, Rizzi M: The structure of Rigidoporus superposed Fo-Fc difference Fourier map, for the T2/T3 active site of mol- lignosus Laccase containing a full complement of copper ecules A and B of LtL. Figure 6 (A) and (B) shows the representations of ions, reveals an asymmetrical arrangement for the T3 cop- the 2Fo-Fc difference Fourier map (cyan colored, the electron density is per pair. J Mol Biol 2004, 342:1519-1531. contoured at 3σ) with a superposed Fo-Fc difference Fourier map (red 21. Enguita FJ, Martins LO, Henriques AO, Carrondo MA: Crystal colored, The electron density is contoured at 3σ), for the T2/T3 active site structure of a bacterial endospore coat component. A laccase with enhanced thermostability properties. J Biol Chem of molecules A and B of LtL respectively. 2003, 278:19416-19425. Click here for file 22. Piontek K, Antorini M, Choinowski T: Crystal structure of a lac- [http://www.biomedcentral.com/content/supplementary/1472- case from the fungus Trametes versicolor at 1.90-A resolu- 6807-7-60-S2.pdf] tion containing a full complement of coppers. J Biol Chem 2002, 277:37663-37669. 23. 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