Structure of the GH9 glucosidase/glucosaminidase from Vibrio cholerae

Glycoside hydrolase family 9 (GH9) of carbohydrate-processing enzymes primarily consists of inverting endoglucanases. A subgroup of GH9 enzymes are believed to act as exo-glucosidases or exo-glucosaminidases, with many being found in organisms of the family Vibrionaceae, where they are proposed to function within the chitin-catabolism pathway. Here, it is shown that the GH9 enzyme from the pathogen Vibrio cholerae (hereafter referred to as VC0615) is active on both chitosan-derived and β-glucoside substrates. The structure of VC0615 at 3.17 A ° resolution is reported from a crystal form with poor diffraction and lattice disorder. VC0615 was highly refractory to crystallization efforts, with crystals only appearing using a high protein concentration under conditions containing the precipitant poly-γ-glutamic acid (PGA). The structure is highly mobile within the crystal lattice, which is likely to reflect steric clashes between symmetry molecules which destabilize crystal packing. The overall tertiary structure of VC0615 is well resolved even at 3.17 A ° resolution, which has allowed the structural basis for the exo-glucosidase/glucosaminidase activity of this enzyme to be investigated

Cloning, purification and characterization of the 6-phospho-3-hexulose isomerase YckF from Bacillus subtilis

The enzyme 6-phospho-3-hexulose isomerase (YckF) from Bacillus subtilis has been prepared and crystallized in a form suitable for X-ray crystallographic analysis. Crystals were grown by the hanging-drop method at 291 K using polyethylene glycol 2000 monomethylether as precipitant. They diffract beyond 1.7 A using an in-house Cu Kalpha source and belong to either space group P6(5)22 or P6(1)22, with unit-cell parameters a = b = 72.4, c = 241.2 A, and have two molecules of YckF in the asymmetric unit

Innovation in Construction Materials-A Review

This study was carried out to review different types of available innovative construction materials. It was found that advancement in nanotechnology, use of mineral admixture, glass and plastic, biological materials, wood and other construction materials have contributed significantly to the growth of discovery and production of innovative construction materials. The implementation of some innovative construction materials, meets the requirements for sustainability, durability, reliability, safety, cost reduction, increasing quality, better mechanical and physical characteristics, flexibility in extreme conditions and locations, simple assembly and environmentally friendly. Construction materials used to carry out project consumed about 40% of the entire cost of the project in the construction industry. The success stories were recorded in the area of turning industrial and agricultural wastes to wealth. This reviewed paper will enrich the database for innovative materials entering the construction industry

Crystallization and preliminary X-ray diffraction analysis of levansucrase (LsdA) from Gluconacetobacter diazotrophicus SRT4

The endophytic bacterium Gluconacetobacter diazotrophicus SRT4 secretes a constitutively expressed levansucrase (LsdA; EC 2.4.1.10), which converts sucrose to fructo-oligosaccharides and levan. Fully active LsdA was purified to high homogeneity by non-denaturing reversed-phase HPLC and was crystallized at room temperature by the hanging-drop vapour-diffusion method using ammonium sulfate and ethanol as precipitants. The crystals are extremely sensitive, but native data have been collected to 2.5 A under cryogenic conditions using synchrotron radiation. LsdA crystals belong to the orthorhombic space group P22(1)2(1) or P2(1)2(1)2, with unit-cell parameters a = 53.80, b = 119.39, c = 215.10 A

Carbohydrate structure: : the rocky road to automation

With the introduction of intuitive graphical software, structural biologists who are not experts in crystallography are now able to build complete protein or nucleic acid models rapidly. In contrast, carbohydrates are in a wholly different situation: scant automation exists, with manual building attempts being sometimes toppled by incorrect dictionaries or refinement problems. Sugars are the most stereochemically complex family of biomolecules and, as pyranose rings, have clear conformational preferences. Despite this, all refinement programs may produce high-energy conformations at medium to low resolution, without any support from the electron density. This problem renders the affected structures unusable in glyco-chemical terms. Bringing structural glycobiology up to ‘protein standards’ will require a total overhaul of the methodology. Time is of the essence, as the community is steadily increasing the production rate of glycoproteins, and electron cryo-microscopy has just started to image them in precisely that resolution range where crystallographic methods falter most

Structural studies of a surface-entropy reduction mutant of O-GlcNAcase

The enzyme O-GlcNAcase catalyses the removal of the O-GlcNAc co/post-translational modification in multicellular eukaryotes. The enzyme has become of acute interest given the intimate role of O-GlcNAcylation in tau modification and stability; small-molecular inhibitors of human O-GlcNAcase are under clinical assessment for the treatment of tauopathies. Given the importance of structure-based and mechanism-based inhibitor design for O-GlcNAcase, it was sought to test whether different crystal forms of the human enzyme could be achieved by surface mutagenesis. Guided by surface-entropy reduction, a Glu602Ala/Glu605Ala variant [on the Gly11-Gln396/Lys535-Tyr715 construct; Roth et al. (2017), Nature Chem. Biol. 13, 610-612] was obtained which led to a new crystal form of the human enzyme. An increase in crystal contacts stabilized disordered regions of the protein, enabling 88% of the structure to be modelled; only 83% was possible for the wild-type construct. Although the binding of the C-terminus was consistent with the wild type, Lys713 in monomer A was bound in the -1 subsite of the symmetry-related monomer A and the active sites of the B monomers were vacant. The new crystal form presents an opportunity for enhanced soaking experiments that are essential to understanding the binding mechanism and substrate specificity of O-GlcNAcase

Evaluation of novel ß-ribosidase substrates for the differentiation of Gram-negative bacteria

Aims:  To synthesize novel substrates for the detection of β-ribosidase and assess their potential for the differentiation of Gram-negative bacteria. Methods and Results:  Two novel chromogenic substrates, 3′,4′-dihydroxyflavone-4′-β-D-ribofuranoside (DHF-riboside) and 5-bromo-4-chloro-3-indolyl-β-D-ribofuranoside (X-riboside) were evaluated along with a known fluorogenic substrate, 4-methylumbelliferyl-β-D-ribofuranoside (4MU-riboside). A total of 543 Gram-negative bacilli were cultured on media containing either DHF-riboside or X-riboside. Hydrolysis of DHF-riboside or X-riboside resulted in the formation of clearly distinguishable black or blue-green colonies, respectively. Hydrolysis of 4MU-riboside was evaluated in a liquid medium in microtiter trays and yielded blue fluorescence on hydrolysis which was measured using fluorimetry. β-Ribosidase activity was widespread with 75% of strains, including 85·6% of Enterobacteriaceae, showing activity with at least one substrate. Genera that demonstrated β-ribosidase activity included Aeromonas, Citrobacter, Enterobacter, Escherichia, Hafnia, Klebsiella, Morganella, Providencia, Pseudomonas, Salmonella and Shigella. In contrast, strains of Proteus spp., Acinetobacter spp., Yersinia enterocolitica, Vibrio cholerae and Vibrio parahaemolyticus generally failed to demonstrate β-ribosidase activity. Conclusions:  The novel substrates DHF-riboside and X-riboside are effective for the detection of β-ribosidase in agar-based media and may be useful for the differentiation and identification of Gram-negative bacteria. Significance and Impact of the Study:  This is the first report describing the application and utility of chromogenic substrates for β-ribosidase. These substrates could be applied in chromogenic media for differentiation of Gram-negative bacteria

The Role of Construction Materials in Building Collapse in Nigeria: A Review

Building failure or collapse happens both in the developed and developing countries but the rate of occurrence in the developing country such as Nigeria is quite alarming and therefore call concern. The menace of building collapse in Nigeria is very alarming and appalling. Building collapse in Nigeria has become the norms they are easily swept under the carpet whenever it occurs. It is rather unfortunate that both human and economic loss we record each time there is a failure or building collapse is rather humongous. There is also a lot of blame game among professional stakeholders in the industry once failure or collapse of building occur and unfortunately, there is no proper scientific investigation in most developing countries such as Nigeria to ascertain the cause of failure or collapse as the case may be in order to avoid any future occurrence. In the cause of this review, it was discovered that building materials such as reinforcing steel, cement, sand, granite, sandcrete blocks and concrete play huge important role in either the collapse or stability of buildings in Nigeria. The roles of these materials are so vital that they contribute immensely to the collapse of buildings. It was concluded that 10-25% of buildings that collapse in Nigeria are as a result of the use of poor quality building materials. The review has extensively dealt with building collapse in relation with the role of building materials during construction

Computational design-of-experiment unveils the conformational reaction coordinate of GH125 α-mannosidases

Conformational analysis of enzyme-catalyzed mannoside hydrolysis has revealed two predominant conformational itineraries through B 2,5 or 3H 4 transitionstate (TS) conformations. A prominent unassigned catalytic itinerary is that of exo-1,6-α-mannosidases belonging to CAZy family 125. A published complex of Clostridium perfringens GH125 enzyme with a nonhydrolyzable 1,6-α-thiomannoside substrate mimic bound across the active site revealed an undistorted 4 C 1 conformation and provided no insight into the catalytic pathway of this enzyme. We show through a purely computational approach (QM/MM metadynamics) that sulfur-for-oxygen substitution in the glycosidic linkage fundamentally alters the energetically accessible conformational space of a thiomannoside when bound within the GH12S active site. Modeling of the conformational free energy landscape (FEL) of a thioglycoside strongly favors a mechanistically uninformative 4 C 1 conformation within the GH125 enzyme active site, but the FEL of corresponding O-glycoside substrate reveals a preference for a Michaelis complex in an oS 2 conformation (consistent with catalysis through a B 2,5 TS). This prediction was tested experimentally by determination of the 3D X-ray structure of the pseudo-Michaelis complex of an inactive (D220N) variant of C. perfringens GH125 enzyme in complex with 1,6-α-mannobiose. This complex revealed unambiguous distortion of the -1 subsite mannoside to an oS 2 conformation, matching that predicted by theory and supporting an oS 2 → B 2,5 → 1S 5 conformational itinerary for GH125 α-mannosidases. This work highlights the power of the QM/MM approach and identified shortcomings in the use of nonhydrolyzable substrate analogues for conformational analysis of enzymebound species