Three-dimensional structures of laccases.

Laccases are phenol oxidases that belong to the family of multi-copper oxidases and the superfamily of cupredoxins. A number of potential industrial applications for laccases have led to intensive structure-function studies and an increased amount of crystal structures has been solved. The objective of this review is to summarize and analyze available crystal structures of laccases. The experimental crystallographic data are now easily available from the websites and electron density maps can be used for the interpretation of the structural models. The crystal structures can give valuable insights into the functional mechanisms and may serve as the basis for the development of laccases for industrial applications.

Three-dimensional structure of cellobiohydrolase II from Trichoderma reesei.

The enzymatic degradation of cellulose is an important process, both ecologically and commercially. The three-dimensional structure of a cellulase, the enzymatic core of CBHII from the fungus Trichoderma reesei reveals an alpha-beta protein with a fold similar to but different from the widely occurring barrel topology first observed in triose phosphate isomerase. The active site of CBHII is located at the carboxyl-terminal end of a parallel beta barrel, in an enclosed tunnel through which the cellulose threads. Two aspartic acid residues, located in the center of the tunnel are the probable catalytic residues.

A near atomic resolution structure of a Melanocarpus albomyces laccase.

We have solved a crystal structure from Melanocarpus albomyces laccase expressed in the filamentous fungus Trichoderma reesei (rMaL) at 1.3A resolution by using synchrotron radiation at 100K. At the moment, this is the highest resolution that has been attained for any multicopper oxidase. The present structure confirmed our earlier proposal regarding the dynamic behaviour of the copper cluster. Thermal ellipsoids of copper atoms indicated movements of trinuclear site coppers. The direction of the type-3 copper motion was perpendicular to the type-2 copper. In addition, the structure at 1.3A resolution allowed us to describe important solvent cavities of the enzyme and the structure is also compared with other known multicopper oxidases. T2 and T3 solvent cavities, and a putative SDS-gate, formed by Ser142, Ser510 and the C-terminal Asp556 of rMaL, are described. We also observed a 2-oxohistidine, an oxidized histidine, possibly caused by a metal-catalysed oxidation by the trinuclear site coppers. To our knowledge, this is the first time that 2-oxohistidine has been observed in a protein crystal structure.

Animal-derived lipocalin allergens exhibit immunoglobulin E cross-reactivity.

BACKGROUND: Although knowledge of the IgE cross-reactivity between allergens is important for understanding the mechanisms of allergy, the regulation of the allergic immune response and the development of efficient modes of allergen immunotherapy, the cross-reactivity of animal allergens is poorly known. OBJECTIVE: The aim of this study was to characterize IgE cross-reactivities between lipocalin proteins, including five animal-derived lipocalin allergens and one human endogenous lipocalin, tear lipocalin (TL). METHODS: The recombinant proteins were validated by chromatography and mass spectrometry. The IgE-binding capacity of the allergens was confirmed by IgE. immunoblotting and IgE immunoblot inhibition. IgE ELISA was performed with sera from 42 atopic patients and 21 control subjects. The IgE cross-reactivities between the lipocalin proteins were determined by ELISA inhibition. RESULTS: ELISA inhibition revealed IgE cross-reactivities between Can f 1 and human TL, between Can f 1 and Can f 2, and between Equ c 1 and Mus m 1. Low levels of IgE to human TL were found in the sera of seven dog-allergic patients of whom six were IgE-positive for Can f 1. CONCLUSION: Several lipocalins exhibited IgE cross-reactivity, probably due to the sequential identity of the proteins and also due to similarities in their three-dimensional structures. The clinical significance of the findings needs to be elucidated. Low-level IgE cross-reactivity can play a role in regulating immune response to lipocalin allergens.

Crystallization and preliminary X-ray analysis of two major xylanases from Trichoderma reesei.

Two major endoxylanases, endo-beta-1,4-xylanase I and II (molecular mass 19 kDa and 21 kDa) from the filamentous fungus Trichoderma reesei have been crystallized by using ammonium sulphate as the precipitating agent. Both crystals were monoclinic and belonged to the space groups C2 (a = 71.9 A, b = 39.0 A, c = 59.9 A, beta = 118.0 degrees, for XYNI) and P2(1) (a = 81.6 A, b = 60.6 A, c = 38.3 A, beta = 94.4, for XYNII). The crystals diffract to at least 2.2 A and 1.5 A, respectively.

Crystallization of the core protein of cellobiohydrolase II from Trichoderma reesei.

Single crystals of the core protein of the cellulase cellobiohydrolase II have been grown in polyethylene glycol 6000 with the hanging drop method. Successful crystallization occurred only when 82 amino acids were removed from the N terminus by papain cleavage. Crystals belong to the space group P2(1) and have cell constants a = 49.1 A, b = 75.8 A, c = 92.9 A, beta = 103.2. The diffraction pattern extends to better than 2.0 A.

Structural comparison of Ntn-hydrolases.

The Ntn-hydrolases (N-terminal nucleophile) are a superfamily of diverse enzymes that has recently been characterized. All of the proteins in this family are activated autocatalytically; they contain an N-terminally located catalytic nucleophile, and they cleave an amide bond. In the present study, the structures of four enzymes of this superfamily are compared in more detail. Although the amino acid sequence homology is almost completely absent, the enzymes share a similar alphabeta betaalpha-core structure. The central beta-sheets in the core were found to have different packing angles, ranging from 5 to 35 degrees. In the Ntn-hydrolases under study, eight totally conserved secondary structure units were found (region C). Five of them were observed to contain the greatest number of conserved and functionally important residues and are therefore crucial for the structure and function of Ntn-hydrolases. Two additional regions, consisting of secondary structure units (regions A and B), were found to be in structurally similar locations, but in different orders in the polypeptide chain. The catalytic machinery is located in the structures in a similar manner, and thus the catalytic mechanisms of all of the enzymes are probably similar. However, the substrate binding and the oxyanion hole differed partially.

Putative helices 3 and 5 of the human vitamin D3 receptor are important for the binding of calcitriol.

We have introduced eleven point mutations into the human vitamin D receptor by site-directed mutagenesis in order to identify some of the amino acid residues that are important for ligand binding. The amino acid residues Ser225, His229, Asp232, Val234, Ser235, Tyr236, Ser237, Lys240, Ile242, Lys246 (helix 3), and Ser275 (helix 5) of the human vitamin D receptor were substituted by alanine. We report here that His229, Asp232, and Ser237 have an important role in the binding of calcitriol. In addition, the amino acid residues Tyr236 and Ser275 also seem to participate in the ligand binding process.

Tryptophan 272: an essential determinant of crystalline cellulose degradation by Trichoderma reesei cellobiohydrolase Cel6A.

Trichoderma reesei cellobiohydrolase Cel6A (formerly CBHII) has a tunnel shaped active site with four internal subsites for the glucose units. We have predicted an additional ring stacking interaction for a sixth glucose moiety with a tryptophan residue (W272) found on the domain surface. Mutagenesis of this residue selectively impairs the enzyme function on crystalline cellulose but not on soluble or amorphous substrates. Our data shows that W272 forms an additional subsite at the entrance of the active site tunnel and suggests it has a specialised role in crystalline cellulose degradation, possibly in guiding a glucan chain into the tunnel.

Structural and functional properties of low molecular weight endo-1,4-beta-xylanases.

There are currently four crystal structures of low molecular weight endo-1,4-beta-xylanases (E.C.3.2.1.8), i.e. family G/11 xylanases, available at the Brookhaven Data Bank: 2 xylanases from Trichoderma reesei (Törrönen et al., 1994; Törrönen and Rouvinen, 1995) and one from Bacillus circulans and another from Trichoderma harzianum (Campbell et al., 1993). They consist of two beta-sheets and one alpha-helix and have been described to resemble a partly-closed right hand. The catalytic residues are two conserved glutamate residues, which are located opposite to each other in an open active site cleft. The catalytic mechanism is thought to resemble that of the widely-studied enzyme lysozyme. The role of one glutamate is to act as an acid/base catalyst whereas the other is a nucleophile and stabilizes the reaction intermediate. Complex structures of partly-bound xylotetraose in mutated XYN from Bacillus circulans (Wakarchuck et al., 1994a) and three recently-obtained structures of XYNII from Trichoderma reesei with epoxyalkyl-xylose derivatives (Havukainen et al., 1996) have provided important information on substrate binding. Family G/11 xylanases show clear amino acid homology and thus have a common fold. However, variations in their functional properties, such as catalytic activity, substrate cleaving patterns, pH optima and thermostabilities, exist.

A combination of weakly stabilizing mutations with a disulfide bridge in the alpha-helix region of Trichoderma reesei endo-1,4-beta-xylanase II increases the thermal stability through synergism.

Thermal stability and other functional properties of Trichoderma reesei endo-1,4-beta-xylanase II (XYNII; family 11) were studied by designed mutations. Mutations at three positions were introduced to the XYNII mutant containing a disulfide bridge (S110C-N154C) in the alpha-helix. The disulfide bridge increased the half-life of XYNII from less than 1 min to 14 min at 65 degrees C. An additional mutation at the C-terminus of the alpha-helix (Q162H or Q162Y) increased the half-life to 63 min. Mutations Q162H and Q162Y alone had a stabilizing effect at 55 degrees C but not at 65 degrees C. The mutations N11D and N38E increased the half-life to about 100 min. Due to the stabilizing mutations the pH stability increased in a wide pH range, but at the same time the activity decreased both in acidic and neutral-alkaline pH, the pH optimum being at pH region 5-6. There was no essential difference between the specific activities of the mutants and the wild-type XYNII.

Vitamin D(3) analogs (MC 1288, KH 1060, EB 1089, GS 1558, and CB 1093): studies on their mechanism of action.

Selected 20-epi and 20-normal vitamin D(3) analogs were studied. First, point mutations were introduced into human vitamin D receptor (VDR) to identify residues important for ligand binding. In helices three, four and five, His229, Asp232, Ser237 and Arg274 seem to have an important role in the binding of calcitriol. Surprisingly, the 20-epi analog MC 1288 did not bind to Ser237. Second, the effects of analogs on VDR degradation were studied. The transcriptionally active 20-epi analogs protected VDR against degradation more efficiently than the 20-normal analogs and calcitriol. With proteasome inhibitor MG-132 formation of Sug-1-RXRbeta-VDR-VDRE complex was detected. The 20-epi analogs effectively prevented its formation. Thus, the 20-epi analogs induce a VDR conformation, which prevents binding of factors mediating VDR degradation. Third, the analogs were found to be powerful regulators of cell cycle progression in MG-63 cells. They arrested cell cycle in the G0/G1 phase at lower concentrations and earlier time points than calcitriol. This was accompanied by hypophosphorylation of Rb followed by strong inhibition of Cdk2 activity. This correlated with increased levels of p27. Cdk2 and cyclin E levels were downregulated but those of p21 and cyclin D1 were not affected. Thus, a similar sequence of events with calcitriol and the analogs in inhibiting MG-63 cell growth was detected but the analogs had much longer lasting and stronger effects than calcitriol. A unifying scheme for the varying effects of vitamin D(3) analogs is presented.

Improved synthesis and characterization of 1,3,4,6-tetra-O-acetyl-2-(N-acetylacetamido)-2-deoxy-beta-D-glucopyranose.

A method from the 1960s to synthesize the N,N-diacetyl derivative of peracetylated beta-D-glucosamine was improved by assistance of molecular sieves. The melting point of the title compound was revised and the structure determined by means of X-ray diffraction.

Structure-function studies of a Melanocarpus albomyces laccase suggest a pathway for oxidation of phenolic compounds.

Melanocarpus albomyces laccase crystals were soaked with 2,6-dimethoxyphenol, a common laccase substrate. Three complex structures from different soaking times were solved. Crystal structures revealed the binding of the original substrate and adducts formed by enzymatic oxidation of the substrate. The dimeric oxidation products were identified by mass spectrometry. In the crystals, a 2,6-dimethoxy-p-benzoquinone and a C-O dimer were observed, whereas a C-C dimer was the main product identified by mass spectrometry. Crystal structures demonstrated that the substrate and/or its oxidation products were bound in the pocket formed by residues Ala191, Pro192, Glu235, Leu363, Phe371, Trp373, Phe427, Leu429, Trp507 and His508. Substrate and adducts were hydrogen-bonded to His508, one of the ligands of type 1 copper. Therefore, this surface-exposed histidine most likely has a role in electron transfer by laccases. Based on our mutagenesis studies, the carboxylic acid residue Glu235 at the bottom of the binding site pocket is also crucial in the oxidation of phenolics. Glu235 may be responsible for the abstraction of a proton from the OH group of the substrate and His508 may extract an electron. In addition, crystal structures revealed a secondary binding site formed through weak dimerization in M. albomyces laccase molecules. This binding site most likely exists only in crystals, when the Phe427 residues are packed against each other.

Structural comparison of two major endo-1,4-xylanases from Trichoderma reesei.

Three-dimensional structures of two major endo-1,4-xylanases, XYNI and XYNII from Trichoderma reesei, have been determined by X-ray crystallography. The amino acid sequences of both enzymes are highly homologous (identity approximately 50%), and both XYNI and XYNII exist as a single domain that contains two mostly antiparallel beta-sheets which are packed against each other. The beta-sheet structure is twisted, forming a cleft where the active site is situated. Two glutamic acids in the cleft, Glu75 and Glu164 in XYNI as well as Glu86 and Glu177 in XYNII, are most likely involved in catalysis. Inspection of the structures reveals that the width of the active site cleft and the number of subsites are different in XYNI and XYNII. The active site is narrower in XYNI and probably contains only three subsites, whereas the number of subsites in XYNII is most likely five. Variations in the surroundings of catalytic residue Glu164XYNI/Glu177XYNII are thought to explain the pH optimum differences observed in XYNI and XYNII.

Analysis of myo-inositol hexakisphosphate hydrolysis by Bacillus phytase: indication of a novel reaction mechanism.

Phytic acid (myo-inositol hexakisphosphate, InsP(6)) hydrolysis by Bacillus phytase (PhyC) was studied. The enzyme hydrolyses only three phosphates from phytic acid. Moreover, the enzyme seems to prefer the hydrolysis of every second phosphate over that of adjacent ones. Furthermore, it is very likely that the enzyme has two alternative pathways for the hydrolysis of phytic acid, resulting in two different myo-inositol trisphosphate end products: Ins(2,4,6)P(3) and Ins(1,3,5)P(3). These results, together with inhibition studies with fluoride, vanadate, substrate and a substrate analogue, indicate a reaction mechanism different from that of other phytases. By combining the data presented in this study with (1) structural information obtained from the crystal structure of Bacillus amyloliquefaciens phytase [Ha, Oh, Shin, Kim, Oh, Kim, Choi and Oh (2000) Nat. Struct. Biol. 7, 147-153], and (2) computer-modelling analyses of enzyme-substrate complexes, a novel mode of phytic acid hydrolysis is proposed.

Three-dimensional structure of the catalytic core of acetylxylan esterase from Trichoderma reesei: insights into the deacetylation mechanism.

Acetylxylan esterase from Trichoderma reesei removes acetyl side groups from xylan. The crystal structure of the catalytic core of the enzyme was solved at 1.9 A resolution. The core has an alpha/beta/alpha sandwich fold, similar to that of homologous acetylxylan esterase from Penicillium purpurogenum and cutinase from Fusarium solani. All three enzymes belong to family 5 of the carbohydrate esterases and the superfamily of the alpha/beta hydrolase fold. Evidently, the enzymes have diverged from a common ancestor and they share the same catalytic mechanism. The catalytic machinery of acetylxylan esterase from T. reesei was studied by comparison with cutinase, the catalytic site of which is well known. Acetylxylan esterase is a pure serine esterase having a catalytic triad (Ser90, His187, and Asp175) and an oxyanion hole (Thr13 N, and Thr13 O gamma). Although the catalytic triad of acetylxylan esterase has been reported previously, there has been no mention of the oxyanion hole. A model for the binding of substrates is presented on the basis of the docking of xylose. Acetylxylan esterase from T. reesei is able to deacetylate both mono- and double-acetylated residues, but it is not able to remove acetyl groups located close to large side groups such as 4-O-methylglucuronic acid. If the xylopyranoside residue is double-acetylated, both acetyl groups are removed by the catalytic triad: first one acetyl group is removed and then the residue is reorientated so that the nucleophilic oxygen of serine can attack the second acetyl group.

Three-dimensional structure of endo-1,4-beta-xylanase II from Trichoderma reesei: two conformational states in the active site.

The three-dimensional structure of endo-1,4-beta-xylanase II (XYNII) from Trichoderma reesei has been determined by X-ray diffraction techniques and refined to a conventional R-factor of 18.3% at 1.8 A resolution. The 190 amino acid length protein was found to exist as a single domain where the main chain folds to form two mostly antiparallel beta-sheets, which are packed against each other in parallel. The beta-sheet structure is twisted, forming a large cleft on one side of the molecule. The structure of XYNII resembles that of Bacillus 1,3-1,4-beta-glucanase. The cleft is an obvious suggestion for an active site, which has putative binding sites for at least four xylose residues. The catalytic residues are apparently the two glutamic acid residues (Glu86 and Glu177) in the middle of the cleft. One structure was determined at pH 5.0, corresponding to the pH optimum of XYNII. The second structure was determined at pH 6.5, where enzyme activity is reduced considerably. A clear structural change was observed, especially in the position of the side chain of Glu177. The observed conformational change is probably important for the mechanism of catalysis in XYNII.

Inversion of the roles of the nucleophile and acid/base catalysts in the covalent binding of epoxyalkyl xyloside inhibitor to the catalytic glutamates of endo-1,4-beta-xylanase (XYNII): a molecular dynamics study.

X-Ray crystal structures have revealed that 2, 3-epoxypropyl-beta-D-xyloside reacts with endo-1,4-beta-xylanase (XYNII) by forming a covalent bond with Glu86. In contrast, 3, 4-epoxybutyl-beta-D-xyloside forms a covalent bond with Glu177. In the normal enzyme reaction Glu86 acts as the catalytic nucleophile and Glu177 as the acid/base catalyst. To rationalize the observed reactivity of the two mechanism-based inhibitors, we carried out eight 300 ps molecular dynamics simulations for different enzyme-inhibitor complexes. Simulations were done for both stereo isomers (R and S) of the inhibitors and for enzyme in which the protonation state of the nucleophile and acid/base catalyst was normal (Glu86 charged, Glu177 neutral) and in which the roles of the catalytic residues were reversed (Glu86 neutral, Glu177 charged). The number of reactive conformations found in each simulation was used to predict the reactivity of epoxy inhibitors. The conformation was considered to be a reactive one when at the same time (i) the proton of the catalytic acid was close (<2.9/3.4/3.9 A) to the oxirane oxygen of the inhibitor, (ii) the nucleophile was close to the terminal carbon of the oxirane group (<3.4/3.9/4.4 A) and (iii) the nucleophile approached the terminal carbon from a reactive angle (<30/45/60 degrees from an ideal attack angle). On the basis of the number of reactive conformations, 2,3-epoxypropyl-beta-D-xyloside was predicted to form a covalent bond with Glu86 and 3, 4-epoxybutyl-beta-D-xyloside with Glu177, both in agreement with the experiment. Thus, the MD simulations and the X-ray structures indicate that in the covalent binding of 3, 4-epoxybutyl-beta-D-xyloside the roles of the catalytic glutamates of XYNII are reversed from that of the normal enzyme reaction.

Crystallization and preliminary X-ray diffraction studies of the catalytic core of acetyl xylan esterase from Trichoderma reesei.

Acetyl xylan esterase is involved in the biodegradation of hemicellulose. It cleaves O-acetyl groups from xylan, which is the most abundant hemicellulose in nature. The catalytic core of acetyl xylan esterase from T. reesei has been crystallized and X-ray diffraction data at 2.3 A collected. The crystal belongs to the triclinic space group P1 with unit-cell parameters a = 50.3, b = 62. 1, c = 40.0 A, alpha = 110.1, beta = 113.6 and gamma = 97.9 degrees. The asymmetric unit contains two molecules.