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.

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.

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.

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.

Search for the determinants of allergenicity in proteins of the lipocalin family.

Three different lines of analysis have been applied to approach the problem of the allergenicity of certain proteins: biological functions, molecular structures and immunological properties. It is immediately obvious that these three are interdependent. The lipocalin family of proteins includes a significant number of allergens. A considerable amount of data is already available of lipocalins and some insights about allergenic determinants can now be presented. However, more information on the molecular structures and immunological parameters of lipocalin allergens is required.

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.

Thermostability of endo-1,4-beta-xylanase II from Trichoderma reesei studied by electrospray ionization Fourier-transform ion cyclotron resonance MS, hydrogen/deuterium-exchange reactions and dynamic light scattering.

Endo-1,4-beta-xylanase II (XYNII) from Trichoderma reesei is a 21 kDa enzyme that catalyses the hydrolysis of xylan, the major plant hemicellulose. It has various applications in the paper, food and feed industries. Previous thermostability studies have revealed a significant decrease in enzymic activity of the protein at elevated temperatures in citrate buffer [Tenkanen, Puls and Poutanen (1992) Enzyme Microb. Technol. 14, 566-574]. Here, thermostability of XYNII was investigated using both conventional and nanoelectrospray ionization Fourier-transform ion cyclotron resonance MS and hydrogen/deuterium (H/D)-exchange reactions. In addition, dynamic light scattering (DLS) was used as a comparative method to observe possible changes in both tertiary and quaternary structures of the protein. We observed a significant irreversible conformational change and dimerization when the protein was exposed to heat. H/D exchange revealed two distinct monomeric protein populations in a narrow transition temperature region. The conformational change in both the water and buffered solutions occurred in the same temperature region where enzymic-activity loss had previously been observed. Approx. 10-30% of the protein was specifically dimerized when exposed to the heat treatment. However, adding methanol to the solution markedly lowered the transition temperature of conformational change as well as increased the dimerization up to 90%. DLS studies in water confirmed the change in conformation observed by electrospray ionization MS. We propose that the conformational change is responsible for the loss of enzymic activity at temperatures over 50 degrees C and that the functioning of the active site in the enzyme is unfeasible in a new, more labile solution conformation.

Molecular pathogenesis of a disease: structural consequences of aspartylglucosaminuria mutations.

A deficiency of functional aspartylglucosaminidase (AGA) causes a lysosomal storage disease, aspartylglucosaminuria (AGU). The recessively inherited disease is enriched in the Finnish population, where 98% of AGU alleles contain one founder mutation, AGU(Fin). Elsewhere in the world, we and others have described 18 different sporadic AGU mutations. Many of these are predicted to interfere with the complex intracellular maturation and processing of the AGA polypeptide. Proper initial folding of AGA in the endoplasmic reticulum (ER) is dependent on intramolecular disulfide bridge formation and dimerization of two precursor polypeptides. The subsequent activation of AGA occurs autocatalytically in the ER and the protein is transported via the Golgi to the lysosomal compartment using the mannose-6-phosphate receptor pathway. Here we use the three-dimensional structure of AGA to predict structural consequences of AGU mutations, including six novel mutations, and make an effort to characterize every known disease mutation by dissecting the effect of mutations on intracellular stability, maturation, transport and the activity of AGA. Most mutations are substitutions replacing the original amino acid with a bulkier residue. Mutations of the dimer interface prevent dimerization in the ER, whereas active site mutations not only destroy the activity but also affect maturation of the precursor. Depending on their effects on the AGA polypeptide the mutations can be categorized as mild, moderate or severe. These data contribute to the expanding body of knowledge pertaining to molecular pathogenesis of AGU.

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.

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.

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.

The crystal structure of beta-glucosidase from Bacillus circulans sp. alkalophilus: ability to form long polymeric assemblies.

Family 1 of glycosyl hydrolases is a large and biologically important group of enzymes. A new three-dimensional structure of this family, beta-glucosidase from Bacillus circulans sp. alkalophilus is reported here. This is the first structure of beta-glucosidase from an alkaliphilic organism. The model was determined by the molecular replacement method and refined to a resolution of 2.7 A. The quaternary structure of B. circulans sp. alkalophilus beta-glucosidase is an octamer and subunits of the octamer show a similar (beta/alpha)(8) barrel fold to that previously reported for other family 1 enzymes. The crystal structure suggested that Cys169 in the active site is substituted. The Cys169 is located near the putative acid/base catalyst Glu166 and it may contribute to the high pH optimum of the enzyme. The crystal structure also revealed that the asymmetric unit contains two octamers which have a clear binding interaction with each other. The ability of the octamers to link with each other suggested that beta-glucosidase from Bacillus circulans sp. alkalophilus is able to form long polymeric assemblies, at least in the crystalline state.

X-ray studies of recombinant anti-testosterone Fab fragments: the use of PEG 3350 in crystallization.

Recombinant anti-testosterone wild-type Fab fragment and mutant Fab fragments with high binding selectivity developed by protein engineering have been crystallized with and without ligands. Crystals of these Fab fragments were obtained by the vapour-diffusion technique at room temperature using solutions of PEG 3350 with various biological buffers and with a wide pH range. So far, five data sets have been collected from crystals of three Fab-antigen complexes and from two uncomplexed Fab fragments, with resolutions ranging from 2.10 to 3.1 A. Crystallization conditions for Fab fragments were found by using modifications of the low ionic strength PEG 3350 series. Suitable concentrations of PEG 400, MPD and glycerol solutions for use as cryoprotectants in PEG 3350 solutions have been determined. One useful observation was that PEG 3350 is able to work alone as a cryoprotectant. The screening protocol used requires a smaller amount of protein material to achieve auspicious pre-crystals than previously. Results support the claim that PEG 3350 is more suitable for the crystallization of Fab fragments than higher molecular weight PEGs.

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.

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.

The importance of the putative helices 4 and 5 of human vitamin D(3) receptor for conformation and ligand binding.

The 3-D structure of the human vitamin D(3) receptor has not been solved to date. To study the conformation of the ligand binding pocket and the amino acid residues important for binding of calcitriol and its synthetic 20-epi analog MC1288, we have introduced several point mutations into putative helices 4 and 5 of human vitamin D(3) receptor by site-directed mutagenesis. The amino acid residues Ser256, Glu257, Asp258, Gln259, Lys264, Ser265, Ser266, Glu269, Arg274, Ser278, and Phe279 were substituted by alanine. Our results suggest that Arg274 is important for the binding of calcitriol and probably also for the binding of the synthetic vitamin D analog MC1288, whereas Asp258, Gln259, Glu269, and Phe279 may have an important role in stabilizing the conformation of hVDR after ligand binding.

Probing the molecular basis of allergy. three-dimensional structure of the bovine lipocalin allergen Bos d 2.

The three-dimensional structure of the major bovine allergen Bos d 2 has been determined by using x-ray diffraction at 1.8-A resolution. Structurally Bos d 2 is a member of the lipocalin family comprising proteins with transport functions. There is a flat small cavity inside the Bos d 2 protein core suitable for ligand binding, and it is possible that Glu115 and Asn37 inside the core are able to make hydrogen bonds with the ligand. Many allergens from different animals belong to the lipocalin family. The amino acid residue similarities between these lipocalins indicate putative regions for IgE binding. Comparison with the available allergen structures from other sources suggests that these allergens are roughly the same size and that their shape is more spherical than elliptical.

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.

The role of the T-box for the function of the vitamin D receptor on different types of response elements.

The nuclear hormone 1alpha,25-dihydroxyvitamin D3(VD) mainly functions through a heterodimer formed between the VD receptor (VDR) and the retinoid X receptor (RXR). This transcription factor complex specifically recognizes DNA sequences, referred to as VD response elements (VDREs), that are formed by two hexameric core binding motifs arranged either as direct repeats spaced by 3 nt (DR3) or inverted palindromes with nine intervening nucleotides (IP9). Gel shift clipping assays provided the first evidence that VDR-RXR heterodimers form different conformations on these two types of VDREs. Since the T-box within the C-terminal extension of the receptor DNA binding domain (DBD) was previously shown to form a dimerization interface with the partner receptor DBD when bound to DR-type response elements, all six amino acid residues of the VDR T-box were investigated for their role in VDR-RXR heterodimer complex formation on DR3- and IP9-type VDREs. Interestingly, the residue Phe93 (F93) was found to be critical on both types of VDREs, whereas the role of the residue Ile94 (I94) was found to depend on ionic strength of the binding reaction and the nature of the VDRE. However, under physiological conditions I94 was also shown to be critical on both VDRE types. The monitored differences between the two VDR-containing protein-DNA complexes helps in an understanding of the differential action of the nuclear hormone VD and its therapeutically important analogues.