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Point-of-care ultrasound (PoCUS) has high sensitivity and specificity in diagnosing uncomplicated colonic diverticulitis in Western patients. Evidence regarding the accuracy of PoCUS in Asian patients in which diverticulitis frequently occurs in the right-side colon is lacking. This multicenter, 10-y study was aimed at evaluating the diagnostic accuracy of PoCUS in various locations of uncomplicated diverticulitis among Asians. A convenience sample of patients with suspected colonic diverticulitis who had undergone computed tomography (CT) were eligible. Patients undergoing PoCUS before CT were included. The primary outcome was the diagnostic accuracy of PoCUS in the various locations, compared with the final diagnosis made by the expert physicians. The sensitivity, specificity, positive predictive value and negative predictive value were computed. The logistic regression model was used to investigate the possible factors related to the accuracy of PoCUS. A total of 326 patients were included. The overall accuracy of PoCUS was 92% (95% confidence interval [CI]: 89.1%–95.0%) and was lower in the cecum (84.3%, 95% CI: 77.8%–90.8%), compared with other locations (p < 0.0001). Nine of 10 false positives had the final diagnosis of appendicitis: 5 had an outpouching structure whose origin in the cecum could not be traced and 4 had elongated "diverticula." Moreover, body mass index was negatively associated with the accuracy of PoCUS in cecal diverticulitis (odds ratio: 0.79, 95% CI: 0.64–0.97) after adjusting for other covariates. Point-of-care ultrasound exhibits high diagnostic accuracy in diagnosing uncomplicated diverticulitis in the Asian population. However, the accuracy varies according to location, and was relatively low in the cecum. [ABSTRACT FROM AUTHOR]

Objectives: This study aims to investigate the characteristics of patients after free falls at the Level-I trauma centers. The factors associated with survival were differentiated.Methods: This retrospective study was conducted at the National Taiwan University Hospital, the Hsin-Chu branch, and the Yun-Lin branch, all accredited as Level-I trauma centers between January 2010 and September 2020. Adult patients with falls from height of more than one story (i.e. 3.6 m) were included. Clinical data were obtained from electronic medical records. Odds ratios (OR) were computed with 95% confidence intervals (CIs) for significant parameters for survival.Results: A total of 371 patients were included. Only 2 survived to discharge with poor neurologic outcomes in 101 patients with OHCA. The overall mortality rate was 98% and 11% in patients with and without OHCA. A higher falling height with a one-meter increase (OR, 1.14, 95% CI, 1.10-1.19) was significantly related to OHCA, especially the height over 6 m (OR, 3.07, 95% CI, 1.19-7.94). A higher trauma injury severity score (TRISS) was significantly related to survival among patients without OHCA (OR, 1.07, 95% CI, 1.04-1.11), especially TRISS≧0.945 (OR, 5.21, 95% CI, 1.28-21.24). Patients without severe head/neck injury of Abbreviated Injury Scale (AIS)≧3 (OR, 0.17, 95% CI, 0.07-0.42) were positively associated with survivors among patients without OHCA.Conclusion: Patients with traumatic OHCA following falls had a high mortality rate of 98% and dismal outcomes, compared with non-traumatic OHCA. Falling heights, especially over 6 m was associated with OHCA. Patients without OHCA had a mortality rate of 11%. Patients with a higher TRISS, especially more than 0.945, or without severe head injury had more chances to survive in the non-OHCA group. The study provided the evidence to guide termination of high futility resuscitation for traumatic OHCA secondary to falls to conserve the clinical resources. [ABSTRACT FROM AUTHOR]

Global precipitation is becoming increasingly intense due to the extreme climate. Therefore, creating new technology to manage water resources is crucial. To create a sustainable urban and ecological environment, a water level and water quality control system implementing artificial intelligence is presented in this research. The proposed smart monitoring system consists of four sensors (two different liquid level sensors, a turbidity and pH sensor, and a water oxygen sensor), a control module (an MCU, a motor, a pump, and a drain), and a power and communication system (a solar panel, a battery, and a wireless communication module). The system focuses on low-cost Internet of Things (IoT) devices along with low power consumption and high precision. This proposal collects rainfall from the preceding 10 years in the application region as well as the region's meteorological bureau's weekly weather report and uses artificial intelligence to compute the appropriate water level. More importantly, the adoption of dynamic adjustment systems can reserve and modify water resources in the application region more efficiently. Compared to existing technologies, the measurement approach utilized in this study not only achieves cost savings exceeding 60% but also enhances water level measurement accuracy by over 15% through the successful implementation of water level calibration decisions utilizing multiple distinct sensors. Of greater significance, the dynamic adjustment systems proposed in this research offer the potential for conserving water resources by more than 15% in an effective manner. As a result, the adoption of this technology may efficiently reserve and distribute water resources for smart cities as well as reduce substantial losses caused by anomalous water resources, such as floods, droughts, and ecological concerns. [ABSTRACT FROM AUTHOR]

Abstract: The thermostable 1,3–1,4-β-glucanase PtLic16A from the fungus Paecilomyces thermophila catalyzes stringent hydrolysis of barley β-glucan and lichenan with an outstanding efficiency and has great potential for broad industrial applications. Here, we report the crystal structures of PtLic16A and an inactive mutant E113A in ligand-free form and in complex with the ligands cellobiose, cellotetraose and glucotriose at 1.80Å to 2.25Å resolution. PtLic16A adopts a typical β-jellyroll fold with a curved surface and the concave face forms an extended ligand binding cleft. These structures suggest that PtLic16A might carry out the hydrolysis via retaining mechanism with E113 and E118 serving as the nucleophile and general acid/base, respectively. Interestingly, in the structure of E113A/1,3–1,4-β-glucotriose complex, the sugar bound to the −1 subsite adopts an intermediate-like (α-anomeric) configuration. By combining all crystal structures solved here, a comprehensive binding mode for a substrate is proposed. These findings not only help understand the 1,3–1,4-β-glucanase catalytic mechanism but also provide a basis for further enzymatic engineering. [Copyright &y& Elsevier]

Periodic ground via lattice is investigated to suppress the propagation of parallel-plate mode between two ground planes in a multiple layer package or printed circuit board. This parallel-plate mode (or ground/ground mode) plays a main role in the noise coupling or electromagnetic interference of the power planes or signal traces embedded between two ground planes. An analytical solution of the stopband cutoff frequency for designing the ground via pitch and diameter is derived, based on the eigenfunctions expansion method of the cavity model. Using the ground via lattice method and the analytical design solution, coupling noise mitigation and radiation emission reduction of power planes embedded between ground planes are demonstrated numerically and experimentally. Another example is the design of embedded planar electromagnetic bandgap (EBG) structure. The etched power plane of planar EBG structure embedded between the ground planes does not have a bandgap due to the coupling of the ground/ground mode. The minimum ground via number is designed to reproduce the bandgap of the embedded planar EBG structures by using the proposed analytical solution. [ABSTRACT FROM PUBLISHER]

Xylan-1,4-ß-xylosidase (ß-xylosidase) hydrolyses xylooligomers at their non-reducing ends into individual xylose units. Recently, XylC, a ß-xylosidase from Thermoanaerobacterium saccharolyticum JW/SL-YS485, was found to be structurally different from corresponding glycosyl hydrolases in the CAZy database (http://www.cazy.org/), and was subsequently classified as the first member of a novel family of glycoside hydrolases (GH120). In the present paper, we report three crystal structures of XylC in complex with Tris, xylobiose and xylose at 1.48-2.05 Å (1 Å=0.1 nm) resolution. XylC assembles into a tetramer, and each monomer comprises two distinct domains. The core domain is a right-handed parallel ß-helix (residues 1-75 and 201-638) and the flanking region (residues 76-200) folds into a ß-sandwich domain. The enzyme contains an open carbohydrate-binding cleft, allowing accommodation of longer xylo-oligosaccharides. On the basis of the crystal structures and in agreement with previous kinetic data, we propose that XylC cleaves the glycosidic bond by the retaining mechanism using two acidic residues Asp382 (nucleophile) and Glu405 (general acid/base). In addition to the active site, nine other xylose-binding sites were consistently observed in each of the four monomers, providing a possible reason for the high tolerance of product inhibition. [ABSTRACT FROM AUTHOR]

Abstract: The pectate lyase (Bsp165PelA) from Bacillus sp. N16-5 has great potential in industrial applications because it shows high specific activity under extremely alkaline conditions. Besides, activity measurement of Bsp165PelA does not require addition of calcium, in a way different from the other pectate lyases. Here we report crystal structures of Bsp165PelA in apo-form and in complex with trigalacturonate. The parallel β-helix, active site residues and substrate binding cleft are similar to those in the other pectate lyases from Polysaccharide Lyase family 1. However, some of the highly conserved Ca2+ binding residues and secondary structures are altered in Bsp165PelA, making it difficult to coordinate with Ca2+ as in the other pectate lyases. We found Bsp165PelA forms some direct enzyme–substrate interactions instead of using Ca2+ ions bridging in the extremely alkaline environment. [Copyright &y& Elsevier]

Abstract: The hyperthermophilic endoglucanase Cel5A from Thermotoga maritima can find applications in lignocellulosic biofuel production, because it catalyzes the hydrolysis of glucan- and mannan-based polysaccharides. Here, we report the crystal structures in apo-form and in complex with three ligands, cellotetraose, cellobiose and mannotriose, at 1.29Å to 2.40Å resolution. The open carbohydrate-binding cavity which can accommodate oligosaccharide substrates with extensively branched chains explained the dual specificity of the enzyme. Combining our structural information and the previous kinetic data, it is suggested that this enzyme prefers β-glucosyl and β-mannosyl moieties at the reducing end and uses two conserved catalytic residues, E253 (nucleophile) and E136 (general acid/base), to hydrolyze the glycosidic bonds. Moreover, our results also suggest that the wide spectrum of Tm_Cel5A substrates might be due to the lack of steric hindrance around the C2-hydroxyl group of the glucose or mannose unit from active-site residues. [Copyright &y& Elsevier]

Synchrotron protein crystallography (PX) remains an indispensable tool for uncovering the atomic structures of biomolecules. TLS 15A1, operated by the protein diffraction group of NSRRC, has been accessible to both academic and industrial users since January 2013. This beamline is energy‐tunable within a range between 5.6 and 15.5 keV, which is suitable for phasing experiments. Over the past 11 years, TLS 15A1 has supported 657 projects and facilitated 1523 experiments across 41,656 h of beamtime. This article describes the beamline layout, studies of factors influencing data quality, and significant scientific contributions. [ABSTRACT FROM AUTHOR]

Recently, with the vigorous development of the Internet of Things (IoT) technology, all kinds of intelligent home appliances in the market are constantly innovating. The public requirements for residential safety and convenience are also increasing. Meanwhile, with the improvement of indigenous medical technology and quality of life, people's average lifespan is gradually increasing. However, countries around the world are facing the problem of aging societies. Hand gesture recognition is gaining popularity in the fields of gesture control, robotics, or medical applications. Therefore, how to create a convenient and smart control system of home appliances for the elderly or the disabled has become the objective of this study. It aims to use Google MediaPipe to develop a hand tracking system, which detected 21 key points of a hand through the camera lens of a mobile device and used a vector formula to calculate the angle of the intersection of two lines based on four key points. After the angle of bending finger is obtained, users' hand gesture can be recognized. Our experiments have confirmed that the recognition precision and recall values of hand gesture for numbers 0–9 reached 98.80% and 97.67%, respectively; and the recognition results were used to control home appliances through the low-cost IoT-Enabled system. [ABSTRACT FROM AUTHOR]

Synchrotron protein crystallography (PX) is one of the most powerful and dominant tools for determining the three‐dimensional structures of biological macromolecules. At the National Synchrotron Radiation Research Center, TLS 13B1 is the first dedicated energy‐tunable and highly automated PX beamline for efficiently solving de novo protein structures in Taiwan and has been open for users since September 2005. With remote access, user support services, and annual PX training courses over 17 years, the number of user groups at TLS 13B1 has grown from 11 to 139. In total, 1,429 projects were supported, and 2090 experiments were hosted during 67,846 hr of beamtime. This paper describes the photon source, beamline components, experimental station equipment, beamline software, beamline application process, and results from our users. [ABSTRACT FROM AUTHOR]

The Sso7c4 from Sulfolobus solfataricus forms a dimer, which is believed to function as a chromosomal protein involved in genomic DNA compaction and gene regulation. Here, we present the crystal structure of wild-type Sso7c4 at a high resolution of 1.63 Å, showing that the two basic C-termini are disordered. Based on the fluorescence polarization (FP) binding assay, two arginine pairs, R11/R22′ and R11′/R22, on the top surface participate in binding DNA. As shown in electron microscopy (EM) images, wild-type Sso7c4 compacts DNA through bridging and bending interactions, whereas the binding of C-terminally truncated proteins rigidifies and opens DNA molecules, and no compaction of the DNA occurs. Moreover, the FP, EM and fluorescence resonance energy transfer (FRET) data indicated that the two basic and flexible C-terminal arms of the Sso7c4 dimer play a crucial role in binding and bending DNA. Sso7c4 has been classified as a repressor-like protein because of its similarity to Escherichia coli Ecrep 6.8 and Ecrep 7.3 as well as Agrobacterium tumefaciens ACCR in amino acid sequence. Based on these data, we proposed a model of the Sso7c4-DNA complex using a curved DNA molecule in the catabolite activator protein-DNA complex. The DNA end-to-end distance measured with FRET upon wild-type Sso7c4 binding is almost equal to the distance measured in the model, which supports the fidelity of the proposed model. The FRET data also confirm the EM observation showing that the binding of wild-type Sso7c4 reduces the DNA length while the C-terminal truncation does not. A functional role for Sso7c4 in the organization of chromosomal DNA and/or the regulation of gene expression through bridging and bending interactions is suggested. [ABSTRACT FROM AUTHOR]

β-Mannanase has found various biotechnological applications because it is capable of degrading mannans into smaller sugar components. A highly potent example is the thermophilic β-mannanase from Aspergillus niger BK01 (ManBK), which can be efficiently expressed in industrial yeast strains and is thus an attractive candidate for commercial utilizations. In order to understand the molecular mechanism, which helps in strategies to improve the enzyme's performance that would meet industrial demands, 3D-structural information is a great asset. Here, we present the 1.57Å crystal structure of ManBK. The protein adopts a typical (β/α)8 fold that resembles the other GH5 family members. Polysaccharides were subsequently modeled into the substrate binding groove to identify the residues and structural features that may be involved in the catalytic reaction. Based on the structure, rational design was conducted to engineer ManBK in an attempt to enhance its enzymatic activity. Among the 23 mutants that we constructed, the most promising Y216W showed an 18±2.7% increase in specific activity by comparison with the wild type enzyme. The optimal temperature and heat tolerance profiles of Y216W were similar to those of the wild type, manifesting a preserved thermostability. Kinetic studies showed that Y216W has higher kcat values than the wild type enzyme, suggesting a faster turnover rate of catalysis. In this study we applied rational design to ManBK by using its crystal structure as a basis and identified the Y216W mutant that shows great potentials in industrial applications. [ABSTRACT FROM AUTHOR]

Highlights: [•] This is the structure of GH131 family enzyme in complex with polysaccharide. [•] The broadened substrate binding cleft may explain the substrate diversity. [•] The hydrolysis of GH131 family enzyme may perform via an inverting mechanism. [ABSTRACT FROM AUTHOR]

S-Adenosylhomocysteine hydrolase (SAHH) catalyzes the reversible conversion of S-adenosylhomocysteine into adenosine and homocysteine. The SAHH from Thermotoga maritima ( TmSAHH) was expressed in Escherichia coli and the recombinant protein was purified and crystallized. TmSAHH crystals belonging to space group C2, with unit-cell parameters a = 106.3, b = 112.0, c = 164.9 Å, β = 103.5°, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.85 Å resolution. Initial phase determination by molecular replacement clearly indicated that the crystal contains one homotetramer per asymmetric unit. Further refinement of the crystal structure is in progress. [ABSTRACT FROM AUTHOR]

The β-L-arabinofuranosidase (HypBA1) from Bifidobacterium longum JCM 1217 hydrolyzes the β-1,2-linked arabinofuranose disaccharide to release L-arabinoses. HypBA1 was classified into glycoside hydrolase family 127 (GH127) by the CAZy website (). The enzyme was expressed in Escherichia coli and the purified recombinant protein was crystallized. Crystals belonging to the primitive hexagonal space group P3 x21, with unit-cell parameters a = b = 75.9, c = 254.0 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.78 Å resolution. A BLASTP search () of the Protein Data Bank did not reveal any similar crystal structures. Structural determination by using SeMet MAD and MIR methods is in progress. [ABSTRACT FROM AUTHOR]

• A lever viscoelastic wall (LVEW) that has VE and friction dampers in a device was proposed. • The LVEW amplifies the frame lateral displacement and damper force to the frame structure. • Three full-scale wall specimens were tested to validate the cyclic response. • An iterative procedure was proposed to obtain the displacement amplification for VE dampers. This paper presents a new steel wind- and seismic-resisting wall, called a lever viscoelastic wall (LVEW) that includes a velocity-dependent VE damper and a displacement-dependent friction damper in a device, characterized by two different motion phases. In Phase one motion (i.e., small drifts), the VE damper which is activated by a lever delivers amplified lateral force and energy dissipation to the frame structure. In Phase two motion (i.e., medium-to-large drifts), the VE damper is constrained by a stopper and the friction damper, instead, is activated to provide frictional energy dissipation to the frame. The damping is provided by incorporating VE material and sliding friction in a wall configuration, reducing both the wind and earthquake responses of structures in a single unit. The objectives of the work were to (1) develop the force transfer mechanism and kinematics of the LVEW, (2) verify the cyclic response of LVEWs at low and large drifts, and (3) evaluate the limitation of LVEWs for the applications. Three full-scale LVEW specimens were designed and tested to validate the hysteretic response in two motion phases. The hysteretic response of LVEWs obtained from the test was compared to that from the analytical formulation. [ABSTRACT FROM AUTHOR]

The structure of MoeN5, a unique prenyltransferase involved in the biosynthesis of the antibiotic moenomycin, is reported. MoeN5 catalyzes the reaction of geranyl diphosphate (GPP) with the cis-farnesyl group in phosphoglycolipid 5 to form the (C25) moenocinyl-sidechain-containing lipid 7. GPP binds to an allylic site (S1) and aligns well with known S1 inhibitors. Alkyl glycosides, glycolipids, can bind to both S1 and a second site, S2. Long sidechains in S2 are 'bent' and co-locate with the homoallylic substrate isopentenyl diphosphate in other prenyltransferases. These observations support a MoeN5 mechanism in which 5 binds to S2 with its C6-C11 group poised to attack C1 in GPP to form the moenocinyl sidechain, with the more distal regions of 5 aligning with the distal glucose in decyl maltoside. The results are of general interest because they provide the first structures of MoeN5 and a structural basis for its mechanism of action, results that will facilitate the design of new antibiotics. [ABSTRACT FROM AUTHOR]

The structure of MoeN5, a unique prenyltransferase involved in the biosynthesis of the antibiotic moenomycin, is reported. MoeN5 catalyzes the reaction of geranyl diphosphate (GPP) with the cis-farnesyl group in phosphoglycolipid 5 to form the (C25) moenocinyl-sidechain-containing lipid 7. GPP binds to an allylic site (S1) and aligns well with known S1 inhibitors. Alkyl glycosides, glycolipids, can bind to both S1 and a second site, S2. Long sidechains in S2 are 'bent' and co-locate with the homoallylic substrate isopentenyl diphosphate in other prenyltransferases. These observations support a MoeN5 mechanism in which 5 binds to S2 with its C6-C11 group poised to attack C1 in GPP to form the moenocinyl sidechain, with the more distal regions of 5 aligning with the distal glucose in decyl maltoside. The results are of general interest because they provide the first structures of MoeN5 and a structural basis for its mechanism of action, results that will facilitate the design of new antibiotics. [ABSTRACT FROM AUTHOR]

Omalizumab, an anti-IgE antibody, used to treat severe allergic asthma and chronic idiopathic urticaria, binds to IgE in blood or membrane-bound on B lymphocytes but not to IgE bound to its high (FcεRI) or low (CD23) affinity receptor. Mutagenesis studies indicate overlapping FcεRI and omalizumab-binding sites in the Cε3 domain, but crystallographic studies show FcεRI and CD23-binding sites that are far apart, so how can omalizumab block IgE from binding both receptors? We report a 2.42-Å omalizumab-Fab structure, a docked IgE-Fc/omalizumab-Fab structure consistent with available experimental data, and the free energy contributions of IgE residues to binding omalizumab, CD23, and FcεRI. These results provide a structural and physical basis as to why omalizumab cannot bind receptor-bound IgE and why omalizumab-bound IgE cannot bind to CD23/FcεRI. They reveal the key IgE residues and their roles in binding omalizumab, CD23, and FcεRI. [ABSTRACT FROM AUTHOR]

S-adenosylhomocysteine (SAH) hydrolase catalyzes the reversible hydrolysis of SAH into adenosine and homocysteine by using NAD + as a cofactor. The enzyme from Thermotoga maritima (tmSAHH) has great potentials in industrial applications because of its hyperthermophilic properties. Here, two crystal structures of tmSAHH in complex with NAD + show both open and closed conformations despite the absence of bound substrate. Each subunit of the tetrameric enzyme is composed of three domains, namely the catalytic domain, the NAD + -binding domain and the C-terminal domain. The NAD + binding mode is clearly observed and a substrate analogue can also be modeled into the active site, where two cysteine residues in mesophilic enzymes are replaced by serine and threonine in tmSAHH. Notably, the C-terminal domain of tmSAHH lacks the second loop region of mesophilic SAHH, which is important in NAD + binding, and thus exposes the bound cofactor to the solvent. The difference explains the higher NAD + requirement of tmSAHH because of the reduced affinity. Furthermore, the feature of missing loop is consistently observed in thermophilic bacterial and archaeal SAHHs, and may be related to their thermostability. [ABSTRACT FROM AUTHOR]

A thermophilic glycoside hydrolase family 16 (GH16) β-1,3-1,4-glucanase from Clostridium thermocellum (CtLic16A) holds great potentials in industrial applications due to its high specific activity and outstanding thermostability. In order to understand its molecular machinery, the crystal structure of CtLic16A was determined to 1.95 Å resolution. The enzyme folds into a classic GH16 β-jellyroll architecture which consists of two β-sheets atop each other, with the substrate-binding cleft lying on the concave side of the inner β-sheet. Two Bis–Tris propane molecules were found in the positive and negative substrate binding sites. Structural analysis suggests that the major differences between the CtLic16A and other GH16 β-1,3-1,4-glucanase structures occur at the protein exterior. Furthermore, the high catalytic efficacy and thermal profile of the CtLic16A are preserved in the enzyme produced in Pichia pastoris , encouraging its further commercial applications. [ABSTRACT FROM AUTHOR]

Wereport the results of an investigation of the activity of aseries of amidine and bisamidine compounds against Staphylococcus aureusand Escherichiacoli. The most active compounds bound to an AT-richDNA dodecamer (CGCGAATTCGCG)2and using DSC were foundto increase the melting transition by up to 24 °C. Several compoundsalso inhibited undecaprenyl diphosphate synthase (UPPS) with IC50values of 100–500 nM, and we found good correlations(R2= 0.89, S. aureus; R2= 0.79, E. coli) between experimental and predicted cell growth inhibition by usingDNA ΔTmand UPPS IC50experimental results together with one computed descriptor. We alsosolved the structures of three bisamidines binding to DNA as wellas three UPPS structures. Overall, the results are of general interestin the context of the development of resistance-resistant antibioticsthat involve multitargeting. [ABSTRACT FROM AUTHOR]

Summary YisP is involved in biofilm formation in Bacillus subtilis and has been predicted to produce C30 isoprenoids. We determined the structure of YisP and observed that it adopts the same fold as squalene and dehydrosqualene synthases. However, the first aspartate-rich motif found in essentially all isoprenoid synthases is aspartate poor in YisP and cannot catalyze head-to-head condensation reactions. We find that YisP acts as a phosphatase, catalyzing formation of farnesol from farnesyl diphosphate, and that it is the first phosphatase to adopt the fold seen in the head-to-head prenyl synthases. Farnesol restores biofilm formation in a Δ yisp mutant and modifies lipid membrane structure similarly to the virulence factor staphyloxanthin. This work clarifies the role of YisP in biofilm formation and suggests an intriguing possibility that many of the YisP-like homologs found in other bacteria may also have interesting products and functions. [ABSTRACT FROM AUTHOR]

The glycoside hydrolase 10 (GH10) xylanase from Streptomyces sp. 9 (XynAS9) can operate in a broad range of pH and temperature, and thus is a potential candidate for commercial applications. Recently, we engineered XynAS9 via mutating several residues in accordance with the consensus sequences of GH10 thermophilic xylanases in an attempt to improve the enzyme thermostability and thermotolerance. The most promising effects were observed in the double mutant V81P/G82E. In order to investigate the molecular mechanism of the improved thermal profile of XynAS9, complex crystal structures of the wild type (WT) and mutant (MT) enzyme were solved at 1.88–2.05 Å resolution. The structures reveal a classical GH10 (β/α) 8 TIM-barrel fold. In MT XynAS9, E82 forms several interactions to its neighboring residues, which might aid in stabilizing the local structure. Furthermore, the MT structure showed lower B factors for individual residues compared to the WT structure, reflecting the increased MT protein rigidity. Analyses of the XynAS9 structures also delineate the detailed enzyme–substrate interaction network. More importantly, possible explanations for the enhanced thermal profiles of MT XynAS9 are proposed, which may be a useful strategy for enzyme engineering in the future. [ABSTRACT FROM AUTHOR]

Oxidized polyvinyl alcohol hydrolase (OPH) catalyzes the cleavage of C–C bond in β-diketone. It belongs to the α/β-hydrolase family and contains a unique lid region that covers the active site. The lid is the most variable region when pOPH from Pseudomonas sp. VM15C and sOPH from Sphingopyxis sp. 113P3 are compared. The wild-type enzymes and the pOPH mutants W255A, W255Y and W255F were analyzed for lipase activity by using p-nitrophenyl (pNP) esters as the substrates. The wild-type enzymes showed increased K m and decreased k cat / K m with the acyl chain length, and the mutants showed reduced k cat / K m for pNP acetate, indicating the importance of Trp255 in sequestering the active site from solvent. The significantly lower activity for pNP butyrate can be a result of product inhibition, as suggested by the complex crystal structures, in which butyric acid, DMSO or PEG occupied the same substrate-binding cleft. The mutant activity was retained with pNP caprylate and pNP laurate as the substrates, reflecting the amphipathic nature of the cleft. Moreover, the disulfide bond formation of Cys257/267 is important for the activity of pOPH, but it is not essential for sOPH, which has a shorter lid structure. [ABSTRACT FROM AUTHOR]

Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease. [ABSTRACT FROM AUTHOR]

Octaprenyl pyrophosphate synthase (OPPs), which belongs to the E-type prenyltransferase family, catalyses the successive condensation of farnesyl pyrophosphate with five isopentenyl pyrophosphate molecules to form trans-C40-octaprenyl pyrophosphate (OPP). OPP is essential for the biosynthesis of bacterial ubiquinone or menaquinone side chains, which play an important role in the electron-transport system. Here, Escherichia coli OPPs was expressed, purified and crystallized. The crystals, which belonged to the orthorhombic space group P21212, with unit-cell parameters a = 117.0, b = 128.4, c = 46.4 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.2 Å resolution. Initial phase determination by molecular replacement (MR) clearly indicated that the crystal contained one homodimer per asymmetric unit. Further model building and structural refinement are in progress. [ABSTRACT FROM AUTHOR]

Cellulase 12A from Thermotoga maritima ( TmCel12A) is a hyperthermostable β-1,4-endoglucanase. We recently determined the crystal structures of TmCel12A and its complexes with oligosaccharides. Here, by using site-directed mutagenesis, the role played by Arg60 and Tyr61 in a unique surface loop of TmCel12A was investigated. The results are consistent with the previously observed hydrogen bonding and stacking interactions between these two residues and the substrate. Interestingly, the mutant Y61G had the highest activity when compared with the wild-type enzyme and the other mutants. It also shows a wider range of working temperatures than does the wild type, along with retention of the hyperthermostability. The k and K values of Y61G are both higher than those of the wild type. In conjunction with the crystal structure of Y61G-substrate complex, the kinetic data suggest that the higher endoglucanase activity is probably due to facile dissociation of the cleaved sugar moiety at the reducing end. Additional crystallographic analyses indicate that the insertion and deletion mutations at the Tyr61 site did not affect the overall protein structure, but local perturbations might diminish the substrate-binding strength. It is likely that the catalytic efficiency of TmCel12A is a subtle balance between substrate binding and product release. The activity enhancement by the single mutation of Y61G provides a good example of engineered enzyme for industrial application. [ABSTRACT FROM AUTHOR]

1,3-1,4-β- d-Glucanase has been widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems. Engineering of enzymes for better thermostability is of great importance because it not only can broaden their industrial applications, but also facilitate exploring the mechanism of enzyme stability from structural point of view. To obtain enzyme with higher thermostability and specific activity, structure-based rational design was carried out in this study. Eleven mutants of Fibrobacter succinogenes 1,3-1,4-β- d-glucanase were constructed in attempt to improve the enzyme properties. In particular, the crude proteins expressed in Pichia pastoris were examined firstly to ensure that the protein productions meet the need for industrial fermentation. The crude protein of V18Y mutant showed a 2 °C increment of Tm and W203Y showed ∼30% increment of the specific activity. To further investigate the structure-function relationship, some mutants were expressed and purified from P. pastoris and Escherichia coli. Notably, the specific activity of purified W203Y which was expressed in E. coli was 63% higher than the wild-type protein. The double mutant V18Y/W203Y showed the same increments of Tm and specific activity as the single mutants did. When expressed and purified from E. coli, V18Y/W203Y showed similar pattern of thermostability increment and 75% higher specific activity. Furthermore, the apo-form and substrate complex structures of V18Y/W203Y were solved by X-ray crystallography. Analyzing protein structure of V18Y/W203Y helps elucidate how the mutations could enhance the protein stability and enzyme activity. [ABSTRACT FROM AUTHOR]

The endoglucanase EglA from Piromyces rhizinflata found in cattle stomach belongs to the GH5 family of glycoside hydrolases. The crystal structure of the catalytic domain of EglA shows the (β/α)8-barrel fold typical of GH5 enzymes. Adjacent to the active site of EglA, a loop containing a disulfide bond not found in other similar structures may participate in substrate binding. Because the active site was blocked by the N-terminal His tag of a neighbouring protein molecule in the crystal, enzyme-substrate complexes could not be obtained by soaking but were prepared by cocrystallization. The E154A mutant structure with a cellotriose bound to the −3, −2 and −1 subsites shows an extensive hydrogen-bonding network between the enzyme and the substrate, along with a stacking interaction between Trp44 and the −3 sugar. A possible dimer was observed in the crystal structure, but retention of activity in the E242A mutant suggested that the enzyme probably does not function as a dimer in solution. On the other hand, the first 100 amino acids encoded by the original cDNA fragment are very similar to those in the last third of the (β/α)8-barrel fold, indicating that EglA comprises at least two catalytic domains acting in tandem. [ABSTRACT FROM AUTHOR]

Abstract: Alkaline phytases from Bacillus species, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives to animal feed. The thermostability and neutral optimum pH of Bacillus phytase are attributed largely to the presence of calcium ions. Nonetheless, no report has demonstrated directly how the metal ions coordinate phytase and its substrate to facilitate the catalytic reaction. In this study, the interactions between a phytate analog (myo-inositol hexasulfate) and divalent metal ions in Bacillus subtilis phytase were revealed by the crystal structure at 1.25 Å resolution. We found all, except the first, sulfates on the substrate analog have direct or indirect interactions with amino acid residues in the enzyme active site. The structures also unraveled two active site-associated metal ions that were not explored in earlier studies. Significantly, one metal ion could be crucial to substrate binding. In addition, binding of the fourth sulfate of the substrate analog to the active site appears to be stronger than that of the others. These results indicate that alkaline phytase starts by cleaving the fourth phosphate, instead of the third or the sixth that were proposed earlier. Our high-resolution, structural representation of Bacillus phytase in complex with a substrate analog and divalent metal ions provides new insight into the catalytic mechanism of alkaline phytases in general. [Copyright &y& Elsevier]

β-1,3-1,4-Glucanases catalyze the specific hydrolysis of internal β-1,4-glycosidic bonds adjacent to the 3- O-substituted glucose residues in mixed-linked β-glucans. The thermophilic glycoside hydrolase CtGlu16A from Clostridium thermocellum exhibits superior thermal profiles, high specific activity and broad pH adaptability. Here, the catalytic domain of CtGlu16A was expressed in Escherichia coli, purified and crystallized in the trigonal space group P3121, with unit-cell parameters a = b = 74.5, c = 182.9 Å, by the sitting-drop vapour-diffusion method and diffracted to 1.95 Å resolution. The crystal contains two protein molecules in an asymmetric unit. Further structural determination and refinement are in progress. [ABSTRACT FROM AUTHOR]

The NADH-dependent ( R)-carbonyl reductase from Candida parapsilosis (RCR) catalyzes the asymmetric reduction of 2-hydroxyacetophenone (HAP) to produce ( R)-1-phenyl-1,2-ethanediol [( R)-PED], which is used as a versatile building block for the synthesis of pharmaceuticals and fine chemicals. To gain insight into the catalytic mechanism, the structures of complexes of RCR with ligands, including the coenzyme, are important. Here, the recombinant RCR protein was expressed and purified in Escherichia coli and was crystallized in the presence of NAD+. The crystals, which belonged to the orthorhombic space group P212121, with unit-cell parameters a = 85.64, b = 106.11, c = 145.55 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.15 Å resolution. Initial model building indicates that RCR forms a homotetramer, consistent with previous reports of medium-chain-type alcohol dehydrogenases. [ABSTRACT FROM AUTHOR]

CC-type chemokine ligand 5 (CCL5) has been known to regulate immune responses by mediating the chemotaxis of leukocytes. Depending on the environment, CCL5 forms different orders of oligomers to interact with targets and create functional diversity. A recent CCL5 trimer structure revealed that the N-terminal conversed F12-A13-Y14 (12FAY14) sequence is involved in CCL5 aggregation. The CCL5-12AAA14 mutant with two mutations had a deficiency in the formation of high-order oligomers. In the study, we clarify the respective roles of F12 and Y14 through NMR analysis and structural determination of the CCL5-12AAA14 mutant where F12 is involved in the dimer assembly and Y14 is involved in aggregation. The CCL5-12AAA14 structure contains a unique dimer packing. The backbone pairing shifts for one-residue in the N-terminal interface, when compared to the native CCL5 dimer. This difference creates a new structural orientation and leads to the conclusion that F12 confines the native CCL5 dimer configuration. Without F12 anchoring in the position, the interfacial backbone pairing is permitted to slide. Structural plasticity occurs in the N-terminal interaction. This is the first case to report this structural rearrangement through mutagenesis. The study provides a new idea for chemokine engineering and complements the understanding of CCL5 oligomerization and the role of the 12FAY14 sequence. [ABSTRACT FROM AUTHOR]