Esben Peter Friis

Structural and Mutational Analyses of the Interaction between the Barley alpha-Amylase/Subtilisin Inhibitor and the Subtilisin Savinase Reveal a Novel Mode of Inhibition

Subtilisins represent a large class of microbial serine proteases. To date, there are three-dimensional structures of proteinaceous inhibitors from three families in complex with subtilisins in the Protein Data Bank. All interact with subtilisin via an exposed loop covering six interacting residues. Here we present the crystal structure of the complex between the Bacillus lentus subtilisin Savinase and the barley alpha-amylase/subtilisin inhibitor (BASI). This is the first reported structure of a cereal Kunitz-P family inhibitor in complex with a subtilisin. Structural analysis revealed that BASI inhibits Savinase in a novel way, as the interacting loop is shorter than loops previously reported. Mutational analysis showed that Thr88 is crucial for the inhibition, as it stabilises the interacting loop through intramolecular interactions with the BASI backbone.

Structure and Activity of Paenibacillus Polymyxa Xyloglucanase from Glycoside Hydrolase Family 44.

The enzymatic degradation of plant polysaccharides is emerging as one of the key environmental goals of the early 21st century, impacting on many processes in the textile and detergent industries as well as biomass conversion to biofuels. One of the well known problems with the use of nonstarch (nonfood)-based substrates such as the plant cell wall is that the cellulose fibers are embedded in a network of diverse polysaccharides, including xyloglucan, that renders access difficult. There is therefore increasing interest in the “accessory enzymes,” including xyloglucanases, that may aid biomass degradation through removal of “hemicellulose” polysaccharides. Here, we report the biochemical characterization of the endo-β-1,4-(xylo)glucan hydrolase from Paenibacillus polymyxa with polymeric, oligomeric, and defined chromogenic aryl-oligosaccharide substrates. The enzyme displays an unusual specificity on defined xyloglucan oligosaccharides, cleaving the XXXG-XXXG repeat into XXX and GXXXG. Kinetic analysis on defined oligosaccharides and on aryl-glycosides suggests that both the −4 and +1 subsites show discrimination against xylose-appended glucosides. The three-dimensional structures of PpXG44 have been solved both in apo-form and as a series of ligand complexes that map the −3 to −1 and +1 to +5 subsites of the extended ligand binding cleft. Complex structures are consistent with partial intolerance of xylosides in the −4′ subsites. The atypical specificity of PpXG44 may thus find use in industrial processes involving xyloglucan degradation, such as biomass conversion, or in the emerging exciting applications of defined xyloglucans in food, pharmaceuticals, and cellulose fiber modification.

Three-dimensional structure of a thermophilic family GH11 xylanase from Thermobifida fusca.

Thermostable enzymes employ various structural features dictated at the amino-acid sequence level that allow them to maintain their integrity at higher temperatures. Many hypotheses as to the nature of thermal stability have been proposed, including optimized core hydrophobicity and an increase in charged surface residues to enhance polar solvent interactions for solubility. Here, the three-dimensional structure of the family GH11 xylanase from the moderate thermophile Thermobifida fusca in its trapped covalent glycosyl-enzyme intermediate complex is presented. Interactions with the bound ligand show fewer direct hydrogen bonds from ligand to protein than observed in previous complexes from other species and imply that binding of the xylan substrate involves several water-mediated hydrogen bonds.

Urinary Incontinence and Genital Prolapse: A Prospective Blind Study of the Value of Urodynamic Evaluation

The value of urodynamic examination was studied in 44 women treated for genital prolapse and/or urinary incontinence. A preoperative urodynamic examination was done but not considered in the planning of the operations. The treatment performed after clinical examination was identical to that proposed after urodynamic evaluation in 20 of 34 incontinent patients (59 per cent), while the treatments differed in 14 (41 per cent). The results were considerably better 6 months postoperatively in the former than in the latter patients. Of the 44 patients 11 (25 per cent) had detrusor instability, which resolved 6 months postoperatively in 4 and the symptoms had vanished or decreased in 6.

Analysis of an Industrial Production Suspension of Bacillus Lentus Subtilisin Crystals by Powder Diffraction: A Powerful Quality-Control Tool.

A microcrystalline suspension of Bacillus lentus subtilisin (Savinase) produced during industrial large-scale production was analysed by X-ray powder diffraction (XRPD) and X-ray single-crystal diffraction (MX). XRPD established that the bulk microcrystal sample representative of the entire production suspension corresponded to space group P212121, with unit-cell parameters a = 47.65, b = 62.43, c = 75.74 Å, equivalent to those for a known orthorhombic crystal form (PDB entry 1ndq). MX using synchrotron beamlines at the Diamond Light Source with beam dimensions of 20 × 20 µm was subsequently used to study the largest crystals present in the suspension, with diffraction data being collected from two single crystals (∼20 × 20 × 60 µm) to resolutions of 1.40 and 1.57 Å, respectively. Both structures also belonged to space group P2(1)2(1)2(1), but were quite distinct from the dominant form identified by XRPD, with unit-cell parameters a = 53.04, b = 57.55, c = 71.37 Å and a = 52.72, b = 57.13, c = 65.86 Å, respectively, and refined to R = 10.8% and Rfree = 15.5% and to R = 14.1% and Rfree = 18.0%, respectively. They are also different from any of the forms previously reported in the PDB. A controlled crystallization experiment with a highly purified Savinase sample allowed the growth of single crystals of the form identified by XRPD; their structure was solved and refined to a resolution of 1.17 Å with an R of 9.2% and an Rfree of 11.8%. Thus, there are at least three polymorphs present in the production suspension, albeit with the 1ndq-like microcrystals predominating. It is shown how the two techniques can provide invaluable and complementary information for such a production suspension and it is proposed that XRPD provides an excellent quality-control tool for such suspensions.

Recominant Expression and Immunological Characterization of House Dust Mite Allergen Der P 1

The cysteine protease Der p1 from dust mite of the genus Dermatophagoides pteronyssinus is a major type I allergen. About 80% of house dust mite (HDM) allergic individuals are reactive to this protease in standard assays for detection of IgE. A curative treatment for atopic allergy is immunotherapy (IT) with HDM extracts which are complex mixtures occasionally resulting in anaphylactic reactions. Novozymes focuses on developing a recombinant variant of Der p1 which exhibit lowered risk of IgE‐mediated allergic reactions, while maintaining its ability to trigger proper Th‐cell responses. This may provide a safer alternative for specific IT of HDM allergy. A secreted recombinant form of pro‐Der p 1 expressed by Saccharamyces cerevisiae was obtained by fusion of the pro‐enzyme to a fungal signal peptide. The N‐glycosylation site of Der p1 was mutated resulting in a deglycosylated pro‐enzyme with a molecular mass of 35 kDa. Protein purification procedure was developed to obtain nearly pure Der p1 protein followed by determination of concentration by active‐site‐titration with the cysteine protease inhibitor E64. The deglycosylated recombinant pro‐Der p 1 revealed immunologic similarity to the native Der p 1 molecule when compared in basophile histamine release, IgE‐binding assays and T‐cell proliferation assays. By in silico epitope mapping of a modelled 3‐dimensional structure of Der p1, five putative IgG and IgE epitopes were predicted. By protein engineering, the predicted epitopes were removed one by one in Der p1 and screening for hypoallergenic variants was performed.