Nina Hakulinen

Crystal Structure of a Laccase from Melanocarpus Albomyces with an Intact Trinuclear Copper Site

We have crystallized the ascomycete laccase from Melanocarpus albomyces with all four coppers present and determined the crystal structure at 2.4 Å resolution. The enzyme is heavily glycosylated and consists of three cupredoxin-like domains, similar to those found in the Cu-depleted basidiomycete laccase from Coprinus cinereus. However, there are significant differences in the loops forming the substrate-binding pocket. In addition, the crystal structure of the M. albomyces laccase revealed elongated electron density between all three coppers in the trinuclear copper site, suggesting that an oxygen molecule binds with a novel geometry. This oxygen, required in the reaction, may enter the trinuclear site through the tunnel, which is open in the structure of the C. cinereus laccase. In contrast, the C-terminus on the M. albomyces laccase forms a plug that blocks this access.

Essential role of the C-terminus in Melanocarpus albomyces laccase for enzyme production, catalytic properties and structure.

The C‐terminus of the fungal laccase from Melanocarpus albomyces (MaL) is processed during secretion at a processing site conserved among the ascomycete laccases. The three‐dimensional structure of MaL has been solved as one of the first complete laccase structures. According to the crystal structure of MaL, the four C‐terminal amino acids of the mature protein penetrate into a tunnel leading towards the trinuclear site. The C‐terminal carboxylate group forms a hydrogen bond with a side chain of His140, which also coordinates to the type 3 copper. In order to analyze the role of the processed C‐terminus, site‐directed mutagenesis of the MaL cDNA was performed, and the mutated proteins were expressed in Trichoderma reesei and Saccharomyces cerevisiae. Changes in the C‐terminus of MaL caused major defects in protein production in both expression hosts. The deletion of the last four amino acids dramatically affected the activity of the enzyme, as the deletion mutant delDSGL559 was practically inactive. Detailed characterization of the purified L559A mutant expressed in S. cerevisiae showed the importance of the C‐terminal plug for laccase activity, stability, and kinetics. Moreover, the crystal structure of the L559A mutant expressed in S. cerevisiae showed that the C‐terminal mutation had clearly affected the trinuclear site geometry. The results in this study clearly confirm the critical role of the last amino acids in the C‐terminus of MaL.

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.

Crystal structure of an ascomycete fungal laccase from Thielavia arenaria--common structural features of asco-laccases.

Laccases are copper‐containing enzymes used in various applications, such as textile bleaching. Several crystal structures of laccases from fungi and bacteria are available, but ascomycete types of fungal laccases (asco‐laccases) have been rather unexplored, and to date only the crystal structure of Melanocarpus albomyces laccase (MaL) has been published. We have now solved the crystal structure of another asco‐laccase, from Thielavia arenaria (TaLcc1), at 2.5 Å resolution. The loops near the T1 copper, forming the substrate‐binding pockets of the two asco‐laccases, differ to some extent, and include the amino acid thought to be responsible for catalytic proton transfer, which is Asp in TaLcc1, and Glu in MaL. In addition, the crystal structure of TaLcc1 does not have a chloride attached to the T2 copper, as observed in the crystal structure of MaL. The unique feature of TaLcc1 and MaL as compared with other laccases structures is that, in both structures, the processed C‐terminus blocks the T3 solvent channel leading towards the trinuclear centre, suggesting a common functional role for this conserved ‘C‐terminal plug’. We propose that the asco‐laccases utilize the C‐terminal carboxylic group in proton transfer processes, as has been suggested for Glu498 in the CotA laccase from Bacillus subtilis. The crystal structure of TaLcc1 also shows the formation of a similar weak homodimer, as observed for MaL, that may determine the properties of these asco‐laccases at high protein concentrations.

Structural analysis, enzymatic characterization, and catalytic mechanisms of β‐galactosidase from Bacillus circulans sp. alkalophilus

Crystal structures of native and α‐d‐galactose‐bound Bacillus circulans sp. alkalophilusβ‐galactosidase (Bca‐β‐gal) were determined at 2.40 and 2.25 Å resolutions, respectively. Bca‐β‐gal is a member of family 42 of glycoside hydrolases, and forms a 460 kDa hexameric structure in crystal. The protein consists of three domains, of which the catalytic domain has an (α/β)8 barrel structure with a cluster of sulfur‐rich residues inside the β‐barrel. The shape of the active site is clearly more open compared to the only homologous structure available in the Protein Data Bank. This is due to the number of large differences in the loops that connect the C‐terminal ends of the β‐strands to the N‐terminal ends of the α‐helices within the (α/β)8 barrel. The complex structure shows that galactose binds to the active site as an α‐anomer and induces clear conformational changes in the active site. The implications of α‐d‐galactose binding with respect to the catalytic mechanism are discussed. In addition, we suggest that β‐galactosidases mainly utilize a reverse hydrolysis mechanism for synthesis of galacto‐oligosaccharides.

Crystal structures of Trichoderma reesei β-galactosidase reveal conformational changes in the active site

We have determined the crystal structure of Trichoderma reesei (Hypocrea jecorina) β-galactosidase (Tr-β-gal) at a 1.2Å resolution and its complex structures with galactose, IPTG and PETG at 1.5, 1.75 and 1.4Å resolutions, respectively. Tr-β-gal is a potential enzyme for lactose hydrolysis in the dairy industry and belongs to family 35 of the glycoside hydrolases (GH-35). The high resolution crystal structures of this six-domain enzyme revealed interesting features about the structure of Tr-β-gal. We discovered conformational changes in the two loop regions in the active site, implicating a conformational selection-mechanism for the enzyme. In addition, the Glu200, an acid/base catalyst showed two different conformations which undoubtedly affect the pK(a) value of this residue and the catalytic mechanism. The electron density showed extensive glycosylation, suggesting a structure stabilizing role for glycans. The longest glycan showed an electron density that extends to the eighth monosaccharide unit in the extended chain. The Tr-β-gal structure also showed a well-ordered structure for a unique octaserine motif on the surface loop of the fifth domain.

Determination of thioxylo‐oligosaccharide binding to family 11 xylanases using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry and X‐ray crystallography

Noncovalent binding of thioxylo‐oligosaccharide inhibitors, methyl 4‐thio‐α‐xylobioside (S‐Xyl2‐Me), methyl 4,4II‐dithio‐α‐xylotrioside (S‐Xyl3‐Me), methyl 4,4II,4III‐trithio‐α‐xylotetroside (S‐Xyl4‐Me), and methyl 4,4II,4III,4IV‐tetrathio‐α‐xylopentoside (S‐Xyl5‐Me), to three family 11 endo‐1,4‐β‐xylanases from Trichoderma reesei (TRX I and TRX II) and Chaetomium thermophilum (CTX) was characterized using electrospray ionization Fourier transform ion cyclotron resonance (FT‐ICR) MS and X‐ray crystallography. Ultra‐high mass‐resolving power and mass accuracy inherent to FT‐ICR allowed mass measurements for noncovalent complexes to within |ΔM|average of 2 p.p.m. The binding constants determined by MS titration experiments were in the range 104−103 M−1, decreasing in the series of S‐Xyl5‐Me ≥ S‐Xyl4‐Me > S‐Xyl3‐Me. In contrast, S‐Xyl2‐Me did not bind to any xylanase at the initial concentration of 5–200 µm, indicating increasing affinity with increasing number of xylopyranosyl units, with a minimum requirement of three. The crystal structures of CTX–inhibitor complexes gave interesting insights into the binding. Surprisingly, none of the inhibitors occupied any of the aglycone subsites of the active site. The binding to only the glycone subsites is nonproductive for catalysis, and yet this has also been observed for other family 11 xylanases in complex with β‐d‐xylotetraose [Wakarchuk WW, Campbell RL, Sung WL, Davoodi J & Makoto Y (1994) Protein Sci3, 465–475, and Sabini E, Wilson KS, Danielsen S, Schülein M & Davies GJ (2001) Acta CrystallogrD57, 1344–1347]. Therefore, the role of the aglycone subsites remains controversial despite their obvious contribution to catalysis.

The contribution of polystyrene nanospheres towards the crystallization of proteins.

Background Protein crystallization is a slow process of trial and error and limits the amount of solved protein structures. Search of a universal heterogeneous nucleant is an effort to facilitate crystallizability of proteins. Methodology The effect of polystyrene nanospheres on protein crystallization were tested with three commercial proteins: lysozyme, xylanase, xylose isomerase, and with five research target proteins: hydrophobins HFBI and HFBII, laccase, sarcosine dimethylglycine N-methyltransferase (SDMT), and anti-testosterone Fab fragment 5F2. The use of nanospheres both in screening and as an additive for known crystallization conditions was studied. In screening, the addition of an aqueous solution of nanosphere to the crystallization drop had a significant positive effect on crystallization success in comparison to the control screen. As an additive in hydrophobin crystallization, the nanospheres altered the crystal packing, most likely due to the amphiphilic nature of hydrophobins. In the case of laccase, nanospheres could be used as an alternative for streak-seeding, which insofar had remained the only technique to produce high-diffracting crystals. With methyltransferase SDMT the nanospheres, used also as an additive, produced fewer, larger crystals in less time. Nanospheres, combined with the streak-seeding method, produced single 5F2 Fab crystals in shorter equilibration times. Conclusions All in all, the use of nanospheres in protein crystallization proved to be beneficial, both when screening new crystallization conditions to promote nucleation and when used as an additive to produce better quality crystals, faster. The polystyrene nanospheres are easy to use, commercially available and close to being inert, as even with amphiphilic proteins only the crystal packing is altered and the nanospheres do not interfere with the structure and function of the protein.

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.

Crystallization and preliminary X-ray characterization of Trichoderma reesei hydrophobin HFBII

Hydrophobins are small proteins found in filamentous fungi and characterized by their ability to change the character of a surface by spontaneous self-assembly on a hydrophobic-hydrophilic interface. Hydrophobin HFBII from Trichoderma reesei was crystallized by the hanging-drop vapour-diffusion method at 293 K. Two crystal forms were obtained: a native form and a form crystallized in the presence of manganese chloride. The native crystals were of high symmetry, cubic I23, but only diffracted to 3.25 A. The crystals grown in the presence of manganese were monoclinic and diffracted to 1.0 A with a synchrotron-radiation source. The anomalous difference Patterson map calculated from the home laboratory data showed a strong single peak, possibly caused by manganese present in the crystallization solution.