Lars K. Skov

Characterization of a Coprinus cinereus laccase

A wild-type Coprinus cinereus laccase and its recombinant form expressed in an Aspergillus oryzae host have been purified and characterized. The mature laccase had a molecular mass of 58 kDa by mass spectrometry, an isoelectric point near 4, and two absorption maxima at 278 and 614 nm. Photometric titration with 2,2′-biquinoline showed a Cu/protein(subunit) stoichiometry of ≈4. The electron paramagnetic resonance spectrum showed typical type 1 and type 2 Cu signals, and the circular dichroism showed a typical coordination geometry of the type 1 Cu(II). At pH 5.5, the enzyme had a redox potential of 0.55 V vs. normal hydrogen electrode at its type 1 site. The laccase could oxidize 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) and syringaldazine with optimum pH of 4 and 6.5, respectively. Halides inhibited the laccase. At pH 8.5, the laccase had an optimum temperature between 60°C and 70°C. At the same pH, the laccase had a half-life of >200 or 21.8 min in the presence of 0 or 2 mM H2O2, respectively, at 40°C. Mediated by several phenols and phenothiazines, the laccase was able to oxidatively bleach Direct Blue 1 dye at alkaline pH, making it a promising industrial enzyme candidate.

Crystal structure of the kainate receptor GluR5 ligand-binding core in complex with (S)-glutamate

The X‐ray structure of the ligand‐binding core of the kainate receptor GluR5 (GluR5‐S1S2) in complex with (S)‐glutamate was determined to 1.95 Å resolution. The overall GluR5‐S1S2 structure comprises two domains and is similar to the related AMPA receptor GluR2‐S1S2J. (S)‐glutamate binds as in GluR2‐S1S2J. Distinct features are observed for Ser741, which stabilizes a highly coordinated network of water molecules and forms an interdomain bridge. The GluR5 complex exhibits a high degree of domain closure (26°) relative to apo GluR2‐S1S2J. In addition, GluR5‐S1S2 forms a novel dimer interface with a different arrangement of the two protomers compared to GluR2‐S1S2J.

Structure of recombinant Ves v 2 at 2.0 Å resolution: structural analysis of an allergenic hyaluronidase from wasp venom

Wasp venom from Vespula vulgaris contains three major allergens: Ves v 1, Ves v 2 and Ves v 5. Here, the cloning, expression, biochemical characterization and crystal structure determination of the hyaluronidase Ves v 2 from family 56 of the glycoside hydrolases are reported. The allergen was expressed in Escherichia coli as an insoluble protein and refolded and purified to obtain full enzymatic activity. Three N-glycosylation sites at Asn79, Asn99 and Asn127 were identified in Ves v 2 from a natural source by enzymatic digestions combined with MALDI-TOF mass spectrometry. The crystal structure of recombinant Ves v 2 was determined at 2.0 A resolution and reveals a central (beta/alpha)(7) core that is further stabilized by two disulfide bonds (Cys19-Cys308 and Cys185-Cys197). Based on sequence alignments and structural comparison with the honeybee allergen Api m 2, it is proposed that a conserved cavity near the active site is involved in binding of the substrate. Surface epitopes and putative glycosylation sites have been compared with those of two other major group 2 allergens from Apis mellifera (honeybee) and Dolichovespula maculata (white-faced hornet). The analysis suggests that the harboured allergic IgE-mediated cross-reactivity between Ves v 2 and the allergen from D. maculata is much higher than that between Ves v 2 and the allergen from A. mellifera.

Structure of the house dust mite allergen Der f 2: implications for function and molecular basis of IgE cross-reactivity.

The X‐ray structure of the group 2 major allergen from Dermatophagoides farinae (Der f 2) was determined to 1.83 Å resolution. The overall Der f 2 structure comprises a single domain of immunoglobulin fold with two anti‐parallel β‐sheets. A large hydrophobic cavity is formed in the interior of Der f 2. Structural comparisons to distantly related proteins suggest a role in lipid binding. Immunoglobulin E (IgE) cross‐reactivity between group 2 house dust mite major allergens can be explained by conserved surface areas representing IgE binding epitopes.

Structure-function correlation of intramolecular electron transfer in wild type and single-site mutated azurins

Abstract Intramolecular electron transfer (ET) between the Cys3-Cys26 radical ion (RSSR − ) produced pulse radiolytically and the Cu(II) ion has been studied in four wild type and nine different single site mutants of the blue single-copper protein, azurin. This enabled examination of the rate of this intramolecular ET as a function of driving force and the nature of the medium separating the electron donor and acceptor. Using a tunneling pathway model for ET from donor (RSSR − ) to acceptor (Cu[II]) through a combination of covalent bonds, hydrogen bonds, and space (van der Waals contact) jumps, the electronic coupling decays for protein mediated ET were calculated and potential pathways operating within the different azurins could be predicted. The rates of intramolecular ET and activation parameters for the above azurins correlate well with pathway distance and driving force as predicted by the Marcus theory, using a through-bond ET mechanism.

Increased amylosucrase activity and specificity, and identification of regions important for activity, specificity and stability through molecular evolution

Amylosucrase is a transglycosidase which belongs to family 13 of the glycoside hydrolases and transglycosidases, and catalyses the formation of amylose from sucrose. Its potential use as an industrial tool for the synthesis or modification of polysaccharides is hampered by its low catalytic efficiency on sucrose alone, its low stability and the catalysis of side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling and selective screening (directed evolution) was applied, in order to generate more efficient variants of the enzyme. This resulted in isolation of the most active amylosucrase (Asn387Asp) characterized to date, with a 60% increase in activity and a highly efficient polymerase (Glu227Gly) that produces a longer polymer than the wild‐type enzyme. Furthermore, judged from the screening results, several variants are expected to be improved concerning activity and/or thermostability. Most of the amino acid substitutions observed in the totality of these improved variants are clustered around specific regions. The secondary sucrose‐binding site and β strand 7, connected to the important Asp393 residue, are found to be important for amylosucrase activity, whereas a specific loop in the B‐domain is involved in amylosucrase specificity and stability.

Crystallization and preliminary X‐ray studies of recombinant amylosucrase from Neisseria polysaccharea

Recombinant amylosucrase from Neisseria polysaccharea was crystallized by the vapour-diffusion procedure in the presence of polyethylene glycol 6000. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 95.7, b = 117.2, c = 62.1 A, and diffract to 1.6 A resolution. A p-chloromercuribenzene sulfonate (pcmbs) derivative has been identified and a selenomethionine-substituted protein has been produced and crystallized.

The pH dependence of intramolecular electron transfer in azurins

Abstract Long range electron transfer (LRET) can be induced between the disulfide radical anion, produced pulse radiolytically, and the copper(II) center in the single blue copper protein, azurin. The rate constant of this intramolecular process increases by one order of magnitude upon decreasing the pH from 8 to 4 in all azurins (wild types as well as single site mutants of Pseudomonas aeruginosa azurin) studied so far. In order to pursue the structural basis for the observed pH dependence we have extended our studies to a new mutant, Asp23Ala. In this derivative the aspartate residue is proximal to the electron donating cystine disulfide bridge. However, LRET in this azurin was found to exhibit similar pH dependence as all other wild type and single-site mutants with residues potentially being able to affect the electron transfer process. A detailed consideration of the parameters that determine the efficiency of this process leads to the suggestion that a pH induced change either in the electron transfer distance or in the electronic coupling may be the cause of this behavior.

Maltooligosaccharide disproportionation reaction: an intrinsic property of amylosucrase from Neisseria polysaccharea

Amylosucrase from Neisseria polysaccharea (AS) is a remarkable transglycosidase of family 13 of the glycoside hydrolases that catalyses the synthesis of an amylose‐like polymer from sucrose and is always described as a sucrose‐specific enzyme. Here, we demonstrate for the first time the ability of pure AS to catalyse the disproportionation of maltooligosaccharides by cleaving the α‐1,4 linkage at the non‐reducing end of a maltooligosaccharide donor and transferring the glucosyl unit to the non‐reducing end of another maltooligosaccharide acceptor. Surprisingly, maltose, maltotriose and maltotetraose are very poor glucosyl donors whereas longer maltooligosaccharides are even more efficient glucosyl donors than sucrose. At least five glucose units are required for efficient transglucosylation, suggesting the existence of strong binding subsites, far from the sucrose binding site, at position +4 and above.

Towards the molecular understanding of glycogen elongation by amylosucrase

Amylosucrase from Neisseria polysaccharea (AS) is a transglucosidase from the glycoside‐hydrolase family 13 that catalyzes the synthesis of an amylose‐like polymer from sucrose, without any primer. Its affinity towards glycogen is particularly noteworthy since glycogen is the best D‐glucosyl unit acceptor and the most efficient activator (98‐fold kcat increase) known for this enzyme. Glycogen–enzyme interactions were modeled starting from the crystallographic AS: maltoheptaose complex, where two key oligosaccharide binding sites, OB1 and OB2, were identified. Two maltoheptaose molecules were connected by an α‐1,6 branch by molecular modeling to mimic a glycogen branching. Among the various docking positions obtained, four models were chosen based on geometry and energy criteria. Robotics calculations enabled us to describe a back and forth motion of a hairpin loop of the AS specific B′‐domain, a movement that assists the elongation of glycogen branches. Modeling data combined with site‐directed mutagenesis experiments revealed that the OB2 surface site provides an anchoring platform at the enzyme surface to capture the polymer and direct the branches towards the OB1 acceptor site for elongation. On the basis of the data obtained, a semiprocessive glycogen elongation mechanism can be proposed. Proteins 2007.