Three-dimensional structures of two heavily N-glycosylated Aspergillus sp. family GH3 B-D-glucosidases

The industrial conversion of cellulosic plant biomass into useful products such as biofuels is a major societal goal. These technologies harness diverse plant degrading enzymes, classical exo- and endo-acting cellulases and, increasingly, cellulose-active lytic polysaccharide monooxygenases, to deconstruct the recalcitrant [beta]-D-linked polysaccharide. A major drawback with this process is that the exo-acting cellobiohydrolases suffer from severe inhibition from their cellobiose product. [beta]-D-Glucosidases are therefore important for liberating glucose from cellobiose and thereby relieving limiting product inhibition. Here, the three-dimensional structures of two industrially important family GH3 [beta]-D-glucosidases from Aspergillus fumigatus and A. oryzae, solved by molecular replacement and refined at 1.95 Å resolution, are reported. Both enzymes, which share 78% sequence identity, display a three-domain structure with the catalytic domain at the interface, as originally shown for barley [beta]-D-glucan exohydrolase, the first three-dimensional structure solved from glycoside hydrolase family GH3. Both enzymes show extensive N-glycosylation, with only a few external sites being truncated to a single GlcNAc molecule. Those glycans N-linked to the core of the structure are identified purely as high-mannose trees, and establish multiple hydrogen bonds between their sugar components and adjacent protein side chains. The extensive glycans pose special problems for crystallographic refinement, and new techniques and protocols were developed especially for this work. These protocols ensured that all of the D-pyranosides in the glycosylation trees were modelled in the preferred minimum-energy 4C1 chair conformation and should be of general application to refinements of other crystal structures containing O- or N-glycosylation. The Aspergillus GH3 structures, in light of other recent three-dimensional structures, provide insight into fungal [beta]-D-glucosidases and provide a platform on which to inform and inspire new generations of variant enzymes for industrial application

EMSL and Institute for Integrated Catalysis (IIC) Catalysis Workshop

Within the context of significantly accelerating scientific progress in research areas that address important societal problems, a workshop was held in November 2010 at EMSL to identify specific and topically important areas of research and capability needs in catalysis-related science

Lipid Profile and 5α-Reductase Inhibition Activity of Proprietary Ultrahigh-Pressure Supercritical Carbon Dioxide and Hexane Saw Palmetto Extracts

Inhibition of 5α-reductase (5αR), which blocks the conversion of testosterone to its active metabolite, dihydrotestosterone, has been shown to impact further prostate enlargement (benign prostatic hyperplasia, or BPH). Clinical trials of standardized lipidosterolic extracts of Serenoa repens (LSESr), also known as standardized extracts of saw palmetto, have demonstrated improvement in lower urinary tract symptoms (LUTS) and delayed progression of BPH. The aim of this preclinical study was to compare two standardized LSESr, a proprietary ultrahigh-pressure supercritical carbon dioxide extract of S. repens (UHP-sCESr) and the well-established hexanic extract of S. repens (HESr), for both 5αR inhibition activity and lipid profiles. UHP-sCESr and HESr had nearly identical inhibition curves and comparable IC50 values for 5αR-1 (9.25 ± 0.87 and 9.86 ± 0.11 μg/mL, respectively; p = 0.43) and 5αR-2 (7.47 ± 0.07 and 7.72 ± 0.05 μg/mL, respectively; p = 0.0544). UHP-sCESr and HESr also had comparable lipid profiles based on similar total fatty acid levels (87.7% and 91.5%, respectively), weight/weight comparisons of individual fatty acids, and individual fatty acid ratios to lauric acid. In addition, UHP-sCESr meets the standard set by the United States Pharmacopeia (USP) monograph for authenticity and purity for a supercritical carbon dioxide (SCCO2) extract of saw palmetto, whereas HESr meets the standard set by the European Medicines Agency (EMA) for a well-established medicinal product. In conclusion, based on enzyme inhibition curves and IC50 values, a standardized lipid profile is important to achieve comparable mechanisms of action for lipidosterolic extracts of saw palmetto. UHP-sCESr offers a comparable, standardized LSESr for men with LUTS/BPH in regions where the proprietary HESr is not available

Structure analysis of group I plant nucleases

Anticancer drugs attacking nucleic acids of the target cells have so far been based on animal or fungal ribonucleases. Plant nucleases have been proved to exhibit decreased cytotoxic side effects. Tomato bifunctional nuclease 1 with activity against both single-stranded and double-stranded RNA and DNA was produced in tobacco leaves as recombinant protein. The enzyme crystallizes under several different crystallization conditions. The presence of Zn(2+) ions was confirmed by X-ray fluorescence. First crystallographic data were obtained

Exploration of Methods to Remove Implanted 210^{210}Pb and 210^{210}Po Contamination from Silicon Surfaces

Radioactive contaminants on the surfaces of detector components can be a problematic source of background events for physics experiments searching for rare processes. Exposure to radon is a specific concern because it can result in the relatively long-lived $^{210}$Pb (and progeny) being implanted to significant subsurface depths such that removal is challenging. In this article we present results from a broad exploration of cleaning treatments to remove implanted $^{210}$Pb and $^{210}$Po contamination from silicon, which is an important material used in several rare-event searches. We demonstrate for the first time that heat treatments ("baking") can effectively mitigate such surface contamination, with the results of a 1200 $^{\circ}$C bake consistent with perfect removal. We also report results using wet-chemistry and plasma-based methods, which show that etching can be highly effective provided the etch depth is sufficiently aggressive. Our survey of cleaning methods suggests consideration of multiple approaches during the different phases of detector construction to enable greater flexibility for efficient removal of $^{210}$Pb and $^{210}$Po surface contaminationComment: 8 pages, 7 figure

Structure of the H107R variant of the extracellular domain of mouse NKR P1A at 2.3 A resolution

The structure of the H107R variant of the extracellular domain of the mouse natural killer cell receptor NKR-P1A has been determined by X-ray diffraction at 2.3 Å resolution from a merohedrally twinned crystal. Unlike the structure of the wild-type receptor in space group I4(1)22 with a single chain per asymmetric unit, the crystals of the variant belonged to space group I4(1) with a dimer in the asymmetric unit. Different degrees of merohedral twinning were detected in five data sets collected from different crystals. The mutation does not have a significant impact on the overall structure, but led to the binding of an additional phosphate ion at the interface of the molecules

Organophosphorus acid anhydrolase from Alteromonas macleodii structural study and functional relationship to prolidases.

The bacterial enzyme organophosphorus acid anhydrolase (OPAA) is able to catalyze the hydrolysis of both proline dipeptides (Xaa-Pro) and several types of organophosphate (OP) compounds. The full three-dimensional structure of the manganese-dependent OPAA enzyme is presented for the first time. This enzyme, which was originally isolated from the marine bacterium Alteromonas macleodii, was prepared recombinantly in Escherichia coli. The crystal structure was determined at 1.8 Å resolution in space group C2, with unit-cell parameters a = 133.8, b = 49.2, c = 97.3 Å, β = 125.0°. The enzyme forms dimers and their existence in solution was confirmed by dynamic light scattering and size-exclusion chromatography. The enzyme shares the pita-bread fold of its C-­terminal domain with related prolidases. The binuclear manganese centre is located in the active site within the pita-bread domain. Moreover, an Ni(2+) ion from purification was localized according to anomalous signal. This study presents the full structure of this enzyme with complete surroundings of the active site and provides a critical analysis of its relationship to prolidases