Structural analysis of the GH43 enzyme Xsa43E from Butyrivibrio proteoclasticus

The rumen of dairy cattle can be thought of as a large, stable fermentation vat and as such it houses a large and diverse community of microorganisms. The bacterium Butyrivibrio proteoclasticus is a representative of a significant component of this microbial community. It is a xylan-degrading organism whose genome encodes a large number of open reading frames annotated as fibre-degrading enzymes. This suite of enzymes is essential for the organism to utilize the plant material within the rumen as a fuel source, facilitating its survival in this competitive environment. Xsa43E, a GH43 enzyme from B. proteoclasticus, has been structurally and functionally characterized. Here, the structure of selenomethionine-derived Xsa43E determined to 1.3 Å resolution using single-wavelength anomalous diffraction is reported. Xsa43E possesses the characteristic five-bladed β-propeller domain seen in all GH43 enzymes. GH43 enzymes can have a range of functions, and the functional characterization of Xsa43E shows it to be an arabinofuranosidase capable of cleaving arabinose side chains from short segments of xylan. Full functional and structural characterization of xylan-degrading enzymes will aid in creating an enzyme cocktail that can be used to completely degrade plant material into simple sugars. These molecules have a range of applications as starting materials for many industrial processes, including renewable alternatives to fossil fuels

The three dimensional structure of Bovine Salivary Protein 30b (BSP30b) and its interaction with specific rumen bacteria

Bovine Salivary Protein 30b (BSP30b) is a member of the tubular lipid-binding (TULIP) superfamily that includes the human bactericidal/permeability-increasing proteins (BPI), lipopolysaccharide binding proteins (LBP) and palate, lung, and nasal epithelium carcinoma-associated proteins (PLUNC). BSP30b is most closely related to the PLUNC family and is predominantly found in bovine saliva. There are four BSP30 isoforms (BSP30a-d) and collectively, they are the most abundant protein component of bovine saliva. The PLUNC family members are proposed to be lipid binding proteins, although in most cases their lipid ligands are unknown. Here, we present the X-ray crystal structure of BSP30b at 2.0 Å resolution. We used a double methionine mutant and Se-Met SAD phasing to solve the structure. The structure adopts a curved cylindrical form with a hydrophobic channel formed by an α/β wrap, which is consistent with the TULIP superfamily. The structure of BSP30b in complex with oleic acid is also presented where the ligand is accommodated within the hydrophobic channel. The electron density for oleic acid suggests that the ligand is only partially occupied in the binding site implying that oleic acid may not be the preferred ligand. GFP-tagged BSP30b binds to the surface of olive oil droplets, as observed under fluorescent microscopy, and acts as a surfactant consistent with its association with decreased susceptibility to bloat in cattle. Bacteria extracted directly from bovine rumen contents indicate that the GFP_BSP30b fusion protein binds to a small number of selected bacterial species in vivo. These results suggest that BSP30b may bind to bacterial lipids from specific species and that this abundant protein may have important biological roles via interacting with rumen bacteria during feeding and rumination

Remote access to crystallography beamlines at SSRL: novel tools for training, education and collaboration

The ultimate goal of synchrotron data collection is to obtain the best possible data from the best available crystals, and the combination of automation and remote access at Stanford Synchrotron Radiation Lightsource (SSRL) has revolutionized the way in which scientists achieve this goal. This has also seen a change in the way novice crystallographers are trained in the use of the beamlines, and a wide range of remote tools and hands-on workshops are now offered by SSRL to facilitate the education of the next generation of protein crystallographers