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Multidrug-resistant Acinetobacter baumannii (Ab) has emerged as a leading nosocomial pathogen because of its resistance to most currently available antibiotics. Cystathionine β-lyase (CBL), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the second step in the transsulfuration pathway, which is essential for the metabolic interconversion of the sulfur-containing amino acids homocysteine and methionine. The enzymes of the transsulfuration pathway are considered to be attractive drug targets owing to their specificity to microbes and plants. As a potential target for the development of novel antibacterial drugs, the AbCBL protein was expressed, purified and crystallized. An AbCBL crystal diffracted to 1.57 Å resolution and belonged to the trigonal space group P3112, with unit-cell parameters a = b = 102.9, c = 136.5 Å. The asymmetric unit contained two monomers, with a corresponding VM of 2.3 Å(3) Da(-1) and a solvent content of 46.9%.

Two-metal-dependent sugar isomerases are important in the synthesis of rare sugars. Many of their properties, specifically their metal dependency, have not been sufficiently explored. Here we used X-ray crystallography, site-directed mutagenesis, isothermal titration calorimetry and electron paramagnetic resonance spectroscopy to investigate the molecular determinants of the metal-binding affinity of l-rhamnose isomerase, a two-Mn(2+) -dependent isomerase from Bacillus halodurans (BHRI). The crystal structure of BHRI confirmed the presence of two metal ion-binding sites: a structural metal ion-binding site for substrate binding, and a catalytic metal ion-binding site that catalyzes a hydride shift. One conserved amino acid, W38, in wild-type BHRI was identified as a critical residue for structural Mn(2+) binding and thus the catalytic efficiency of BHRI. This function of W38 was explored by replacing it with other amino acids. Substitution by Phe, His, Lys, Ile or Ala caused complete loss of catalytic activity. The role of W38 was further examined by analyzing the crystal structure of wild-type BHRI and two inactive mutants of BHRI (W38F and W38A) in complex with Mn(2+) . A structural comparison of the mutants and the wild-type revealed differences in their coordination of Mn(2+) , including changes in metal-ligand bond length and affinity for Mn(2+) . The role of W38 was further confirmed in another two-metal-dependent enzyme: xylose isomerase from Bacillus licheniformis. These data suggest that W38 stabilizes protein-metal complexes and in turn assists ligand binding during catalysis in two-metal-dependent isomerases., Structured Digital Abstract: BHRI and BHRI bind by x-ray crystallography (View interaction)., (© 2014 FEBS.)

Sublingual immunotherapy has gained acceptance amongst the paediatric community as it is very well tolerated and is safe. The adverse effects of this therapy is minimal consisting mainly of local side effects within the oral cavity such as itching of the mouth, swelling of the lips and less frequently abdominal pain, wheezing and urticaria has been described. This report is to highlight another local side effect of sublingual immunotherapy which has been observed in 3 of our patients. This is pigmentation of the gums which can occur anytime during the course of the immunotherapy. It resolves on stopping the immunotherapy and is likely due to a local inflammatory process occurring in the gums of these children. There is no associated pain or itching with the pigmentation. It can persist as long as the child is on the immunotherapy.

RNA polymerase (RNAP) plays a crucial role in gene expression in all organisms. It is a multiprotein complex that produces primary transcript RNA. Generally, the basal transcription apparatus in archaea is simpler than the eukaryotic RNA polymerase II counterpart. To understand the structure and function of archaeal RNAP, the TON-0309 gene encoding DNA-directed RNA polymerase subunit L (ToRNAP_L) from Thermococcus onnurineus NA1 was cloned and the protein was overexpressed in Escherichia coli, purified and crystallized. The purified protein was crystallized using the hanging-drop vapour-diffusion method and the crystal diffracted to 2.10 Å resolution. The crystal belonged to the hexagonal space group P6122, with unit-cell parameters a = b = 42.3, c = 211.2 Å. One molecule was present in the asymmetric unit, with a corresponding VM of 2.5 Å(3) Da(-1) and a solvent content of 50.0%.

Along with the co-chaperonin GroES, the chaperonin GroEL plays an essential role in enhancing protein folding or refolding and in protecting proteins against misfolding and aggregation in the cellular environment. The XoGroEL gene (XOO_4288) from Xanthomonas oryzae pv. oryzae was cloned and the protein was expressed, purified and crystallized. The purified XoGroEL protein was crystallized using the hanging-drop vapour-diffusion method and a crystal diffracted to a resolution of 3.4 Å. The crystal belonged to the orthorhombic space group P212121 with 14 monomers in the asymmetric unit, with a corresponding VM of 2.7 Å(3) Da(-1) and a solvent content of 54.5%.

YmfB from Escherichia coli is the Nudix hydrolase involved in the metabolism of thiamine pyrophosphate, an important compound in primary metabolism and a cofactor of many enzymes. In addition, it hydrolyzes (d)NTPs to (d)NMPs and inorganic orthophosphates in a stepwise manner. The structures of YmfB alone and in complex with three sulfates and two manganese ions determined by X-ray crystallography, when compared with the structures of other Nudix hydrolases such as MutT, Ap4Aase and DR1025, provide insight into the unique hydrolysis mechanism of YmfB. Mass-spectrometric analysis confirmed that water attacks the terminal phosphates of GTP and GDP sequentially. Kinetic analysis of binding-site mutants showed that no individual residue is absolutely required for catalytic activity, suggesting that protein residues do not participate in the deprotonation of the attacking water. Thermodynamic integration calculations show that a hydroxyl ion bound to two divalent metal ions attacks the phosphate directly without the help of a nearby catalytic base.

Acinetobacter baumannii causes bacteraemia, pneumonia, other respiratory-tract and urinary-tract infections in humans. OXA-23 carbapenemase-producing A. baumannii K0420859 (A. baumannii OXA-23) is resistant to carbapenem, a common antibacterial drug. To develop an efficient and novel antibacterial drug against A. baumannii OXA-23, D-alanine-D-alanine ligase, which is essential in bacterial cell-wall synthesis, is of interest. Here, the D-alanine-D-alanine ligase (AbDdl) gene from A. baumannii OXA-23 was cloned and expressed, and the AbDdl protein was purified and crystallized; this enzyme can be used as a novel target for an antibacterial drug against A. baumannii OXA-23. The AbDdl crystal diffracted to a resolution of 2.8 Å and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 113.4, b = 116.7, c = 176.5 Å, a corresponding VM of 2.8 Å(3) Da(-1) and a solvent content of 56.3%, and six protomers in the asymmetric unit.

D-Alanine-D-alanine ligase (DDL) catalyzes the biosynthesis of d-alanyl-d-alanine, an essential bacterial peptidoglycan precursor, and is an important drug target for the development of antibacterials. We determined four different crystal structures of DDL from Xanthomonas oryzae pv. oryzae (Xoo) causing Bacteria Blight (BB), which include apo, ADP-bound, ATP-bound, and AMPPNP-bound structures at the resolution between 2.3 and 2.0 Å. Similarly with other DDLs, the active site of XoDDL is formed by three loops from three domains at the center of enzyme. Compared with d-alanyl-d-alanine and ATP-bound TtDDL structure, the γ-phosphate of ATP in XoDDL structure was shifted outside toward solution. We swapped the ω-loop (loop3) of XoDDL with those of Escherichia coli and Helicobacter pylori DDLs, and measured the enzymatic kinetics of wild-type XoDDL and two mutant XoDDLs with the swapped ω-loops. Results showed that the direct interactions between ω-loop and other two loops are essential for the active ATP conformation for D-ala-phosphate formation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Numerous enzymes, such as the pyridoxal 5'-phosphate (PLP)-dependent enzymes, require cofactors for their activities. Using X-ray crystallography, structural snapshots of the L-serine dehydratase catalytic reaction of a bacterial PLP-dependent enzyme were determined. In the structures, the dihedral angle between the pyridine ring and the Schiff-base linkage of PLP varied from 18° to 52°. It is proposed that the organic cofactor PLP directly catalyzes reactions by active conformational changes, and the novel catalytic mechanism involving the PLP cofactor was confirmed by high-level quantum-mechanical calculations. The conformational change was essential for nucleophilic attack of the substrate on PLP, for concerted proton transfer from the substrate to the protein and for directing carbanion formation of the substrate. Over the whole catalytic cycle, the organic cofactor catalyzes a series of reactions, like the enzyme. The conformational change of the PLP cofactor in catalysis serves as a starting point for identifying the previously unknown catalytic roles of organic cofactors.

Platelet-rich plasma (PRP), a blood derivative with high concentrations of platelets, has been found to have high levels of autologous growth factors (GFs), such as transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), fibroblastic growth factor (FGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF). These GFs and other biological active proteins of PRP can promote tissue healing through the regulation of fibrosis and angiogenesis. Moreover, PRP is considered to be safe due to its autologous nature and long-term usage without any reported major complications. Therefore, PRP therapy could be an option in treating overused tendon damage such as chronic tendinopathy. Here, we present a systematic review highlighting the clinical effectiveness of PRP injection therapy in patellar tendinopathy, which is a major cause of athletes to retire from their respective careers.

Images/videos captured from optical devices are usually degraded by turbid media such as haze, smoke, fog, rain and snow. Haze is the most common problem in outdoor scenes because of the atmosphere conditions. This paper proposes a novel single image-based dehazing framework to remove haze artifacts from images, where we propose two novel image priors, called the pixel-based dark channel prior and the pixel-based bright channel prior. Based on the two priors with the haze optical model, we propose to estimate atmospheric light via haze density analysis. We can then estimate transmission map, followed by refining it via the bilateral filter. As a result, high-quality haze-free images can be recovered with lower computational complexity compared with the state-of-the-art approach based on patch-based dark channel prior.

A purified recombinant enzyme from Spirochaeta thermophila, that is suggested to be a cellobiose 2-epimerase, was a 47 kDa monomer with a specific activity of 29.2 U min(-1) for mannobiose. The epimerization activity of the recombinant enzyme for mannobiose was maximal at pH 7.0 and 60 °C with a half-life of 124 h. The enzyme exhibited a higher epimerization activity for mannose or the mannose moiety at the reducing end of β- and α-1,4-glycosyl-mannose than for glucose or the glucose moiety of β- and α-1,4-glycosyl-glucose. The enzyme was identified as a mannobiose 2-epimerase by evaluating its substrate specificity with not only glucose-containing sugars but also mannose-containing sugars. The activities of the reported cellobiose 2-epimerases from Caldicellulosiruptor saccharolyticus, Dictyoglomus turgidum and Ruminococcus marinus for mannobiose were higher than those for cellobiose, strongly suggesting that these enzymes are not cellobiose 2-epimerases but are mannobiose 2-epimerases.

Aminopeptidases are metalloproteinases that degrade N-terminal residues from protein and play important roles in cell growth and development by controlling cell homeostasis and protein maturation. We determined the crystal structure of XoLAP, a leucyl aminopeptidase, at 2.6 Å resolution from Xanthomonas oryzae pv. oryzae, causing the destructive rice disease of bacterial blight. It is the first crystal structure of aminopeptidase from phytopathogens as a drug target. XoLAP existed as a hexamer and the monomer structure consisted of an N-terminal cap domain and a C-terminal peptidase domain with two divalent zinc ions. XoLAP structure was compared with BlLAP and EcLAP (EcPepA) structures. Based on the structural comparison, the molecular model of XoLAP in complex with the natural aminopeptidase inhibitor of microginin FR1 was proposed. The model structure will be useful to develop a novel antibacterial drug against Xoo.

Cellobiose 2-epimerase epimerizes and isomerizes β-1,4- and α-1,4-gluco-oligosaccharides. N-Acyl-D-glucosamine 2-epimerase (DT_epimerase) from Dictyoglomus turgidum has an unusually high catalytic activity towards its substrate cellobiose. DT_epimerase was expressed, purified and crystallized. Crystals were obtained of both His-tagged DT_epimerase and untagged DT_epimerase. The crystals of His-tagged DT_epimerase diffracted to 2.6 Å resolution and belonged to the monoclinic space group P2₁, with unit-cell parameters a=63.9, b=85.1, c=79.8 Å, β=110.8°. With a Matthews coefficient VM of 2.18 Å3 Da(-1), two protomers were expected to be present in the asymmetric unit with a solvent content of 43.74%. The crystals of untagged DT_epimerase diffracted to 1.85 Å resolution and belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a=55.9, b=80.0, c=93.7 Å. One protomer in the asymmetric unit was expected, with a corresponding VM of 2.26 Å3 Da(-1) and a solvent content of 45.6%.

Campylobacter jejuni is one of the major foodborne pathogens causing human infection. Peptide deformylase, a metallohydrolase, catalyzes the deformylation of N-formylated methionine in newly synthesized polypeptides in prokaryotes and some eukaryotic organelles. The deformylation process is an essential step in protein synthesis and has attracted much attention as a potential target for the development of novel antibacterial agents. Here, the cloned codon-optimized def gene from C. jejuni was synthesized and the protein was expressed, purified and crystallized. C. jejuni peptide deformylase crystals obtained at pH 7.0 and pH 6.5 diffracted to 2.9 Å resolution and belonged to the trigonal space group R3, with unit-cell parameters a=b=105.7, c=58.0 Å. One monomer existed in the asymmetric unit, with a corresponding VM of 3.1 Å3 Da(-1) and a solvent content of 60.4%.

Acinetobacter baumannii has received much attention owing to its exceptional ability to develop resistance to currently available antibiotics. Alanine racemase (ALR) catalyzes the racemization of L-alanine to D-alanine with pyridoxal 5'-phosphate (PLP) as a cofactor. The D-alanine product is an essential component of the bacterial cell wall and ALR is a potential target for the development of novel antibacterial drugs. The alr gene from A. baumannii was cloned and the protein (AbALR) was expressed, purified and crystallized. The AbALR crystal diffracted to 2.3 Å resolution and belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 55.1, b = 85.0, c = 167.7 Å. Two protomers were present in the asymmetric unit, with a corresponding V(M) value of 2.3 Å(3) Da(-1) and a solvent content of 47.5%.

Alcohol dehydrogenases (ADHs) are a group of dehydrogenase enzymes that facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of NAD(+) to NADH. In bacteria, some alcohol dehydrogenases catalyze the opposite reaction as part of fermentation to ensure a constant supply of NAD(+). The adh gene from Kangiella koreensis was cloned and the protein (KkADH) was expressed, purified and crystallized. A KkADH crystal diffracted to 2.5 Å resolution and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 94.1, b = 80.9, c = 115.6 Å, β = 111.9°. Four monomers were present in the asymmetric unit, with a corresponding VM of 2.55 Å(3) Da(-1) and a solvent content of 51.8%.

Aldehyde dehydrogenase (ALDH) catalyses the oxidation of aldehydes using NAD(P)(+) as a cofactor. Most aldehydes are toxic at low levels. ALDHs are used to regulate metabolic intermediate aldehydes. The aldh gene from Bacillus cereus was cloned and the ALDH protein was expressed, purified and crystallized. A crystal of the ALDH protein diffracted to 2.6 Å resolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 83.5, b = 93.3, c = 145.5 Å, β = 98.05°. Four protomers were present in the asymmetric unit, with a corresponding VM of 2.55 Å(3) Da(-1) and a solvent content of 51.8%.

RNA-dependent RNA polymerase (RdRp) is essential for the replication of RNA genome-containing positive-strand RNA viruses. We developed a simple colorimetric assay to quantify the RNA synthesis activity of RdRp by measuring the pyrophosphates released during nascent RNA synthesis. RNA polymerase reaction was quenched by heating at 70 °C for 5 min, during which thermostable inorganic pyrophosphatase converted the accumulated pyrophosphates into inorganic phosphates. Subsequently, the amount of inorganic phosphate was measured using a color-developing reagent. Using RdRp's from hepatitis C virus and foot-and-mouth disease virus, we demonstrate that this colorimetric assay facilitates the measurement of RNA polymerase activity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Xo2276 is a putative transcription activator-like effector (TALE) in Xanthomonas oryzae pv. oryzae (Xoo). Xo2276 was expressed with a TAP-tag at the C-terminus in Xoo cells to enable quantitative analysis of protein expression and secretion. Nearly all TAP-tagged Xo2276 existed in an insoluble form; addition of rice leaf extracts from a Xoo-susceptible rice cultivar, Milyang23, significantly stimulated secretion of TAP-tagged Xo2276 into the medium. In a T3SS-defective Xoo mutant strain, secretion of TAP-tagged Xo2276 was blocked. Xo2276 is a Xoo ortholog of Xanthomonas campestris pv. vesicatoria (Xcv) AvrBs3 and contains a conserved DNA-binding domain (DBD), which includes 19.5 tandem repeats of 34 amino acids. Xo2276- DBD was expressed in E. coli and purified. Direct in vitro recognition of Xo2276-DBD on a putative target DNA sequence was confirmed using an electrophoretic mobility shift assay. This is the first study measuring the homologous expression and secretion of Xo2276 in vitro using rice leaf extract and its direct in vitro binding to the specific target DNA sequence.

The genome sequence of Streptomyces coelicolor contains 18 cytochrome P450 enzymes. The recombinant CYP105N1 protein has been expressed in Escherichia coli and purified, and we report the biochemical and structural characterization of CYP105N1 from S. coelicolor. The purified protein exhibited the typical CO-binding spectrum of P450 enzymes and type I binding spectra with estradiol and a coelibactin analog. The oxidation of estradiol by CYP105N1, supported by H(2)O(2), produced estriol. The crystal structure of CYP105N1 was determined at 2.9 Å resolution. An unexpected wide open binding pocket located above the heme group was identified, with a volume of approximately 4299 Å(3). These results suggest that the large open pocket to the active site may be a key feature for easy access of the peptidyl carrier protein-bound substrate to perform the hydroxylation reaction. A molecular docking model with coelibactin showed that the phenyl group of coelibactin is located <4 Å away from the heme-iron, suggesting that CYP105N1 may be involved in the hydroxylation of the phenyl ring of the coelibactin precursor during biosynthesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Etoposide (ETP) treatment of ataxia telangiectasia mutated (ATM) and Rad3-related protein (ATR)-, topoisomerase-binding protein-1 (TopBP1) and human MutY homolog (hMYH)-depleted cells results in a significant reduction in apoptotic signaling. The association between ATR or TopBP1 and hMYH increased following ETP treatment. In hMYH knockdown cells, the interaction between ATR and TopBP1 decreased following ETP treatment. We suggest that hMYH functions as a sensor of ETP-induced apoptosis. The results suggest that in the absence of hMYH, cells are unable to recognize the damage signal and the ATR pathway is not activated.

Cystathionine γ-synthase (CGS) catalyzes the first step in the transsulfuration pathway leading to the formation of cystathionine from O-succinylhomoserine and L-cysteine through a γ-replacement reaction. As an antibacterial drug target against Xanthomonas oryzae pv. oryzae (Xoo), CGS from Xoo (XometB) was cloned, expressed, purified and crystallized. The XometB crystal diffracted to 2.4 Å resolution and belonged to the tetragonal space group I4(1), with unit-cell parameters a=b=165.4, c=241.7 Å. There were four protomers in the asymmetric unit, with a corresponding solvent content of 73.9%.

A recombinant putative N-acyl-D-glucosamine 2-epimerase from Dictyoglomus turgidum was identified as a cellobiose 2-epimerase by evaluating its substrate specificity. The purified enzyme was a 46 kDa monomer with a specific activity of 16.8 μmol min(-1) mg(-1) for cellobiose. The epimerization activity was maximal at pH 7.0 and 70 °C with a half-life of 55 h. The isomerization of the glucose at the reducing end of β-1,4- and α-1,4-linked gluco-oligosaccharides to a fructose moiety by the enzyme took place after the epimerization of the glucose to a mannose moiety. The enzyme converted cellobiose to 12.8 % 4-O-β-D-glucopyranosyl-D-mannose and 54.6 % 4-O-β-D-glucopyranosyl-D-fructose as an equilibrium and converted lactose to 12.8 % epilactose and 54.3 % lactulose.

GIM-1 is a member of the class B carbapenemases (metallo-β-lactamases; MBLs) and has a wide spectrum of activity against carbapenems, penicillins and extended-spectrum cephalosporins, but not aztreonam. GIM-1 presents an enormous challenge to infection control, particularly in the eradication of Gram-negative pathogens including Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii and nonfermenters. There are presently few or no drugs in late-stage development for these pathogens and GIM-1 is a potential target for the development of antimicrobial agents against pathogens producing MBLs. In this study, GIM-1 was cloned, overexpressed and crystallized. The GIM-1 crystals diffracted to 1.4 Å resolution and belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 38.5, b = 67.6, c = 72.8 Å. One molecule is present in the asymmetric unit, with a corresponding V(M) of 1.69 Å(3) Da(-1) and a solvent content of 27.1%.

SFC-1, a class A carbapenemase that confers antibiotic resistance, hydrolyzes the β-lactam rings of β-lactam antibiotics (carbapenems, cephalosporins, penicillins and aztreonam). SFC-1 presents an enormous challenge to infection control, particularly in the eradication of Gram-negative pathogens. As SFC-1 exhibits a remarkably broad substrate range, including β-lactams of all classes, the enzyme is a potential target for the development of antimicrobial agents against pathogens producing carbapenemases. In this study, SFC-1 was cloned, overexpressed, purified and crystallized. The SFC-1 crystal diffracted to 1.6 Å resolution and belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 65.8, b = 68.3, c = 88.8 Å. Two molecules are present in the asymmetric unit, with a corresponding V(M) of 1.99 Å(3) Da(-1) and a solvent content of 38.1%.

Rain removal from a video is a challenging problem and has been recently investigated extensively. Nevertheless, the problem of rain removal from a single image was rarely studied in the literature, where no temporal information among successive images can be exploited, making the problem very challenging. In this paper, we propose a single-image-based rain removal framework via properly formulating rain removal as an image decomposition problem based on morphological component analysis. Instead of directly applying a conventional image decomposition technique, the proposed method first decomposes an image into the low- and high-frequency (HF) parts using a bilateral filter. The HF part is then decomposed into a "rain component" and a "nonrain component" by performing dictionary learning and sparse coding. As a result, the rain component can be successfully removed from the image while preserving most original image details. Experimental results demonstrate the efficacy of the proposed algorithm.

Xanthomonas oryzae pv. oryzae (Xoo) is a plant bacterial pathogen that causes bacterial blight (BB) disease, resulting in serious production losses of rice. The crystal structure of malonyl CoA-acyl carrier protein transacylase (XoMCAT), encoded by the gene fabD (Xoo0880) from Xoo, was determined at 2.3 Å resolution in complex with N-cyclohexyl-2-aminoethansulfonic acid. Malonyl CoA-acyl carrier protein transacylase transfers malonyl group from malonyl CoA to acyl carrier protein (ACP). The transacylation step is essential in fatty acid synthesis. Based on the rationale, XoMCAT has been considered as a target for antibacterial agents against BB. Protein-protein interaction between XoMCAT and ACP was also extensively investigated using computational docking, and the proposed model revealed that ACP bound to the cleft between two XoMCAT subdomains.

Collaborative researches of three-dimensional molecular modeling can be limited by different time zones and locations. A networked virtual environment can be utilized to overcome the problem caused by the temporal and spatial differences. However, traditional approaches did not sufficiently consider integration of different computing environments, which were characterized by types of applications, roles of users, and so on. We propose a collaborative molecular modeling environment to integrate different molecule modeling systems using a virtual tunneling service. We integrated Co-Coot, which is a collaborative crystallographic object-oriented toolkit, with VRMMS, which is a virtual reality molecular modeling system, through a collaborative tunneling system. The proposed system showed reliable quantitative and qualitative results through pilot experiments.

OXA-23, a class D carbapenemase that confers widespread antibiotic resistance hydrolyzes the beta-lactam rings of beta-lactam antibiotics, presenting an enormous challenge to infection control, particularly in the eradication of pathogenic bacteria such as Acinetobacter baumannii, one of six top-priority dangerous pathogens. Thus, the enzyme is a potential target for developing antimicrobial agents against pathogens producing carbapenemases. In this study, OXA-23 was purified and crystallized at 298 K and X-ray diffraction data from OXA-23 crystal were collected at 2.03 A resolution using synchrotron radiation. The crystal of OXA-23 belonged to space group P4(1) with unit cell parameters a = 82.47, b = 82.47 and c = 172.01 A. Analysis of the packing density showed that the asymmetric unit probably contained two molecules with a solvent content of 73.64%.

The proteins in the fatty-acid synthesis pathway in bacteria have significant potential as targets for the development of antibacterial agents. An essential elongation step in fatty-acid synthesis is performed by β-ketoacyl-acyl carrier protein synthase I (FabB). The organism Xanthomonas oryzae pv. oryzae (Xoo) causes a destructive bacterial blight disease of rice. The XoFabB protein from Xoo was expressed, purified and crystallized for the three-dimensional structure determination that is essential for the development of specific inhibitors of the enzyme. An XoFabB crystal diffracted to 3.0 Å resolution and belonged to the tetragonal space group P4(1), with unit-cell parameters a = b = 82.2, c = 233.2 Å. Assuming that the crystallographic structure contains four molecules in the asymmetric unit, the corresponding V(M) would be 2.18 Å(3) Da(-1) and the solvent content would be 43.5%. The initial structure was determined by the MOLREP program with an R factor of 44.0% and does contain four monomers in the asymmetric unit.

Ribosomal protein S3 (rpS3) is a multifunctional protein involved in translation, DNA repair, and apoptosis. The relationship between rpS3 and cyclin-dependent kinases (Cdks) involved in cell cycle regulation is not yet known. Here, we show that rpS3 is phosphorylated by Cdk1 in G2/M phase. Co-immunoprecipitation and GST pull-down assays revealed that Cdk1 interacted with rpS3. An in vitro kinase assay showed that Cdk1 phosphorylated rpS3 protein. Phosphorylation of rpS3 increased in nocodazole-arrested mitotic cells; however, treatment with Cdk1 inhibitor or Cdk1 siRNA significantly attenuated this phosphorylation event. The phosphorylation of a mutant form of rpS3, T221A, was significantly reduced compared with wild-type rpS3. Decreased phosphorylation and nuclear accumulation of T221A was much more pronounced in G2/M phase. These results suggest that the phosphorylation of rpS3 by Cdk1 occurs at Thr221 during G2/M phase and, moreover, that this event is important for nuclear accumulation of rpS3.

Xanthomonas oryzae pv. oryzae (Xoo) produces a putative effector, XoAvrBs2. We expressed XoAvrBs2 homologously in Xoo with a TAP-tag at the C-terminus to enable quantitative analysis of protein expression and secretion. Addition of rice leaf extracts from both Xoo-sensitive and Xoo-resistant rice cultivars to the Xoo cells induced expression of the XoAvrBs2 gene at the transcriptional and translational levels, and also stimulated a remarkable amount of XoAvrBs2 secretion into the medium. In a T3SS-defective Xoo mutant strain, secretion of the TAPtagged XoAvrBs2 was blocked. Thus, we elucidated the transcriptional and translational expressions of the XoAvrBs2 gene in Xoo was induced in vitro by the interaction with rice and the induced secretion of XoAvrBs2 was T3SSdependent. It is the first report to measure the homologous expression and secretion of XoAvrBs2 in vitro by rice leaf extract. Our system for the quantitative analysis of effector protein expression and secretion could be generally used for the study of host-pathogen interactions.

The effect of human MutY homolog (hMYH) on the activation of checkpoint proteins in response to hydroxyurea (HU) and ultraviolet (UV) treatment was investigated in hMYH-disrupted HEK293 cells. hMYH-disrupted cells decreased the phosphorylation of Chk1 upon HU or UV treatment and increased the phosphorylation of Cdk2 and the amount of Cdc25A, but not Cdc25C. In siMYH-transfected cells, the increased rate of phosphorylated Chk1 upon HU or UV treatment was lower than that in siGFP-transfected cells, meaning that hMYH was involved in the activation mechanism of Chk1 upon DNA damage. The phosphorylation of ataxia telangiectasia and Rad3- related protein (ATR) upon HU or UV treatment was decreased in hMYH-disrupted HEK293 and HaCaT cells. Co-immunoprecipitation experiments showed that hMYH was immunoprecipitated by anti-ATR. These results suggest that hMYH may interact with ATR and function as a mediator of Chk1 phosphorylation in response to DNA damage.

Ribose-5-phosphate isomerase (Rpi) catalyzes the conversion of D-ribose 5-phosphate (R5P) to D-ribulose 5-phosphate, which is an important step in the non-oxidative pathway of the pentose phosphate pathway and the Calvin cycle of photosynthesis. Recently, Rpis have been used to produce valuable rare sugars for industrial purposes. Of the Rpis, D-ribose-5-phosphate isomerase B from Clostridium thermocellum (CtRpi) has the fastest reactions kinetics. While Thermotoga maritime Rpi (TmRpi) has the same substrate specificity as CtRpi, the overall activity of CtRpi is approximately 200-fold higher than that of TmRpi. To understand the structural basis of these kinetic differences, we determined the crystal structures, at 2.1-Å resolution or higher, of CtRpi alone and bound to its substrates, R5P, D-ribose, and D-allose. Structural comparisons of CtRpi and TmRpi showed overall conservation of their structures with two notable differences. First, the volume of the CtRpi substrate binding pocket (SBP) was 20% less than that of the TmRpi SBP. Second, the residues next to the sugar-ring opening catalytic residue (His98) were different. We switched the key residues, involved in SBP shaping or catalysis, between CtRpi and TmRpi by site-directed mutagenesis, and studied the enzyme kinetics of the mutants. We found that tight interactions between the two monomers, narrow SBP width, and the residues near the catalytic residue are all critical for the fast enzyme kinetics of CtRpi., (© Springer-Verlag 2011)

Sensitization to perennial aeroallergens correlates with the risk of persistent asthma (AS) in children. In tropical Singapore, multiple codominant species of mites abound in the indoor environment, and preferential species-specific sensitization has been associated with different phenotypes of allergic disease. We investigated the pattern of mite component-specific IgE (mcsIgE) in children with different phenotypes of clinical allergic disease in an environment with multiple mite species exposure. A prospective evaluation of newly diagnosed patients with clinical diagnosis of allergic rhinitis (AR), atopic dermatitis (AD), or AS and sensitization to one or more aeroallergens were performed. Sera were tested for specific IgE against an extensive panel of Dermatophagoides pteronyssinus and Blomia tropicalis allergens. A total of 253 children were included, mean age 7.3 yr, 79% fulfilled criteria for AR, 46% AS, 71% AD, and 31% for all three. Sensitization to one or both mites was observed in 91% of children, 89% were sensitized to D. pteronyssinus, and 70% to B. tropicalis. The most common mite allergens recognized by these atopic children were Der p 1 (64%), Der p 2 (71%), Blo t 5 (45%), Blo t 7 (44%), and Blo t 21 (56%). Specific IgE responses to an increased number of distinct mite allergens correlated with the complexity of the allergic phenotype. In multivariate analysis, an increased risk for the multi-systemic phenotype (AR + AS + AD) was associated with sensitization to an increased repertoire of mite components (three or more) (OR 4.3, 95% CI 2.1-8.8, p = 0.001) and a positive parental history of AS (OR 2.4, 95% CI 1.2-2.9, p = 0.013). A highly pleiomorphic IgE response to the prevalent indoor mites is associated with the presence of a multi-systemic allergic phenotype in childhood in a tropical environment., (© 2011 John Wiley & Sons A/S.)

Whole-genome sequence analysis of Bacillus halodurans ATCC BAA-125 revealed an isomerase gene (rhaA) encoding an L-rhamnose isomerase (L-RhI). The identified L-RhI gene was cloned from B. halodurans and over-expressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,257 bp capable of encoding a polypeptide of 418 amino acid residues with a molecular mass of 48,178 Da. The molecular mass of the purified enzyme was estimated to be ∼48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 121 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had an optimal pH and temperature of 7 and 70°C, respectively, with a k(cat) of 8,971 min⁻¹ and a k(cat)/K(m) of 17 min⁻¹mM⁻¹ for L-rhamnose. Although L-RhIs have been characterized from several other sources, B. halodurans L-RhI is distinguished from other L-RhIs by its high temperature optimum (70°C) with high thermal stability of showing 100% activity for 10 h at 60°C. The half-life of the enzyme was more than 900 min and ∼25 min at 60°C and 70°C, respectively, making B. halodurans L-RhI a good choice for industrial applications. This work describes one of the most thermostable L-RhI characterized thus far.

Background: UDP-glucose dehydrogenase (UGDH) is the sole enzyme that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. The product is used in xenobiotic glucuronidation in hepatocytes and in the production of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers, while inhibition of UGDH diminished tumor angiogenesis in vivo. A better understanding of the conformational changes occurring during the UGDH reaction cycle will pave the way for inhibitor design and potential cancer therapeutics., Methodology: Previously, the substrate-bound of UGDH was determined to be a symmetrical hexamer and this regular symmetry is disrupted on binding the inhibitor, UDP-α-D-xylose. Here, we have solved an alternate crystal structure of human UGDH (hUGDH) in complex with UDP-glucose at 2.8 Å resolution. Surprisingly, the quaternary structure of this substrate-bound protein complex consists of the open homohexamer that was previously observed for inhibitor-bound hUGDH, indicating that this conformation is relevant for deciphering elements of the normal reaction cycle., Conclusion: In all subunits of the present open structure, Thr131 has translocated into the active site occupying the volume vacated by the absent active water and partially disordered NAD+ molecule. This conformation suggests a mechanism by which the enzyme may exchange NADH for NAD+ and repolarize the catalytic water bound to Asp280 while protecting the reaction intermediates. The structure also indicates how the subunits may communicate with each other through two reaction state sensors in this highly cooperative enzyme.

Bacterial blight (BB), a devastating disease caused by Xanthomonas oryzae pv. oryzae (Xoo), causes serious production losses of rice in Asian countries. Protein misfolding may interfere with the function of proteins in all living cells and must be prevented to avoid cellular disaster. All cells naturally contain molecular chaperones that assist the unfolded proteins in folding into the native structure. One of the well characterized chaperone complexes is GroEL-GroES. GroEL, which consists of two chambers, captures misfolded proteins and refolds them. GroES is a co-chaperonin protein that assists the GroEL protein as a lid that temporarily closes the chamber during the folding process. Xoo4289, the GroES gene from Xoo, was cloned and expressed for X-ray crystallographic study. The purified protein (XoGroES) was crystallized using the hanging-drop vapour-diffusion method and a crystal diffracted to 2.0 Å resolution. The crystal belonged to the hexagonal space group P6(1), with unit-cell parameters a=64.4, c=36.5 Å. The crystal contains a single molecule in the asymmetric unit, with a corresponding VM of 2.05 Å3 Da(-1) and a solvent content of 39.9%.

Purpose: We developed a bimolecular fluorescence complementation (BiFC) strategy using Dronpa, a new fluorescent protein with reversible photoswitching activity and fast responsibility to light, to monitor protein-protein interactions in cells., Procedures: Dronpa was split at residue Glu164 in order to generate two Dronpa fragments [Dronpa N-terminal: DN (Met1-Glu164), Dronpa C-terminal: DC (Gly165-Lys224)]. DN or DC was separately fused with C terminus of hHus1 or N terminus of hRad1. Flexible linker [(GGGGS)×2] was introduced to enhance Dronpa complementation by hHus1-hRad1 interaction. Furthermore, we developed expression vectors to visualize the interaction between hMYH and hHus1. Gene fragments corresponding to the coding regions of hMYH and hHus1 were N-terminally or C-terminally fused with DN and DC coding region., Results: Complemented Dronpa fluorescence was only observed in HEK293 cells cotransfected with hHus1-LDN and DCL-hRad1 expression vectors, but not with hHus1-LDN or DCL-hRad1 expression vector alone. Western blot analysis of immunoprecipitated samples using anti-c-myc or anti-flag showed that DN-fused hHus1 interacted with DC-fused hRad1. Complemented Dronpa fluorescence was also observed in cells cotransfected with hMYH-LDN and DCL-hHus1 expression vectors or hMYH-LDN and hHus1-LDC expression vectors. Furthermore, complemented Dronpa, induced by the interaction between hMYH-LDN and DCL-hHus1, showed almost identical photoswitching activity as that of native Dronpa., Conclusion: These results demonstrate that BiFC using Dronpa can be successfully used to investigate protein-protein interaction in live cells. Furthermore, the fact that complemented Dronpa has a reversible photoswitching activity suggests that it can be used as a tool for tracking protein-protein interaction.

L-Rhamnose isomerases catalyze isomerization between L-rhamnose (6-deoxy-L-mannose) and L-rhamnulose (6-deoxy-L-fructose), which is the first step in rhamnose catabolism. L-Rhamnose isomerase from Bacillus halodurans ATCC BAA-125 (BHRI) exhibits interesting characteristics such as high thermostability and selective substrate specificity. BHRI fused with an HHHHHH sequence was purified and crystallized in order to elucidate the molecular basis of its unique enzymatic properties. The crystals were grown by the hanging-drop vapour-diffusion method and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 83.2, b = 164.9, c = 92.0 A, beta = 116.0 degrees . Diffraction data were collected to 2.5 A resolution. According to a Matthews coefficient calculation, there are four monomers in the asymmetric unit with a V(M) of 3.0 A(3) Da(-1) and a solvent content of 59.3%. The initial structure of BHRI has been determined by the molecular-replacement method.

Acetyl-CoA carboxylases (ACCs) have been highlighted as therapeutic targets for obesity and diabetes, as they play crucial roles in fatty acid metabolism. ACC activity is regulated through the short-term mechanism of inactivation by reversible phosphorylation. Here, we report the crystal structures of the biotin carboxylase (BC) domain of human ACC2 phosphorylated by AMP-activated protein kinase (AMPK). The phosphorylated Ser222 binds to the putative dimer interface of BC, disrupting polymerization and providing the molecular mechanism of inactivation by AMPK. We also determined the structure of the human BC domain in complex with soraphen A, a macrocyclic polyketide natural product. This structure shows that the compound binds to the binding site of phosphorylated Ser222, implying that its inhibition mechanism is the same as that of phosphorylation by AMPK., (Copyright 2009 Elsevier Inc. All rights reserved.)

DNA gyrase is a type II topoisomerase that is essential for chromosome segregation and cell division owing to its ability to modify the topological forms of bacterial DNA. In this study, the N-terminal fragment of the GyrB subunit of DNA gyrase from Xanthomonas oryzae pv. oryzae was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.10 A resolution using a synchrotron-radiation source. The crystal belonged to space group I4(1), with unit-cell parameters a = b = 110.27, c = 70.75 A. The asymmetric unit contained one molecule, with a V(M) of 2.57 A(3) Da(-1) and a solvent content of 50.2%.

Rare sugars are used for many industrial and medical purposes and are produced by the interconversion between aldoses and ketoses catalyzed by sugar and sugar-phosphate isomerases. Recently, Clostridium thermocellum d-ribose-5-phosphate isomerase (CTRPI), an aldose-ketose isomerase, was cloned in order to synthesize d-allose and its substrate specificity was further characterized for industrial usage. CTRPI has a novel substrate specificity that differs from those of other isomerases, which have broad substrate specificities. CTRPI prefers aldose substrates such as l-talose, d-ribose and d-allose. CTRPI was purified and crystallized in order to determine its three-dimensional structure and thus to elucidate its enzymatic reaction mechanism and understand its substrate specificity. The crystal belonged to the trigonal space group P3(2)21, with unit-cell parameters a = b = 69.5, c = 154.4 angstrom, and diffracted to 1.9 angstrom resolution. According to Matthews coefficient calculations, the crystallographic structure consists of a dimer in the asymmetric unit, with a V(M) of 3.2 angstrom(3) Da(-1) and a solvent content of 61.7%.

Rad9-Rad1-Hus1 (9-1-1) is a checkpoint protein complex playing roles in DNA damage sensing, cell cycle arrest, DNA repair or apoptosis. Human 8-oxoguanine DNA glycosylase (hOGG1) is the major DNA glycosylase responsible for repairing a specific aberrantly oxidized nucleotide, 7,8-dihydro-8-oxoguanine (8-oxoG). In this study, we identified a novel interaction between hOGG1 and human 9-1-1, and investigated the functional consequences of this interaction. Co-immunoprecipitation assays using transiently transfected HEK293 cells demonstrated an interaction between hOGG1 and the 9-1-1 proteins. Subsequently, GST pull-down assays using bacterially expressed and purified hOGG1-His and GST-fused 9-1-1 subunits (GST-hRad9, GST-hRad1, and GST-hHus1) demonstrated that hOGG1 interacted directly with the individual subunits of the human 9-1-1 complex. In vitro excision assay, which employed a DNA duplex containing an 8-oxoG/C mismatch, showed that hRad9, hRad1, and hHus1 enhanced the 8-oxoG excision and beta-elimination activities of hOGG1. In addition, the presence of hRad9, hRad1, and hHus1 enhanced the formation of covalently cross-linked hOGG1-8-oxoG/C duplex complexes, as determined by a trapping assay using NaBH(4). A trimeric human 9-1-1 complex was purified from Escherichia coli cell transformed with hRad9, His-fused hRad1, or His-fused hHus1 expressing vectors. It also showed the similar activity to enhance in vitro hOGG1 glycosylase activity, compared with individual human 9-1-1 subunits. Detection of 8-oxoG in HEK293 cells using flow cytometric and spectrofluorometric analysis revealed that over-expression of hOGG1 or human 9-1-1 reduced the formation of 8-oxoG residues following the H(2)O(2) treatment. The highest 8-oxoG reduction was observed in HEK293 cells over-expressing hOGG1 and all the three subunits of human 9-1-1. These indicate that individual human 9-1-1 subunits and human 9-1-1 complex showed almost the same abilities to enhance the in vitro 8-oxoG excision activity of hOGG1, but that the greatest effect to remove 8-oxoG residues in H(2)O(2)-treated cells was derived from the 9-1-1 complex as a whole.

Xanthomonas oryzae pv. oryzae (Xoo) causes the serious disease bacterial blight in rice. The pepA (Xoo0834) gene from Xoo is one of around 100 genes that have been selected for the design of antibacterial drugs. The pepA gene encodes leucine aminopeptidase (LAP), an exopeptidase that catalyzes the hydrolysis of leucine residues from the N-terminus of a protein or peptide. This enzyme was expressed in Escherichia coli, purified and crystallized, and preliminary X-ray structural studies have been carried out. The LAP crystal diffracted to 2.6 A resolution and belonged to the cubic space group P2(1)3. The unit-cell volume of the crystal was compatible with the presence of two monomers in the asymmetric unit.

The bacterial beta-ketoacyl-ACP synthase III (KASIII) encoded by the gene fabH (Xoo4209) from Xanthomonas oryzae pv. oryzae, a plant pathogen, is an important enzyme in the elongation steps of fatty-acid biosynthesis. It is expected to be one of the enzymes responsible for bacterial blight (BB), a serious disease that results in huge production losses of rice. As it represents an important target for the development of new antibacterial drugs against BB, determination of the crystal structure of the KAS III enzyme is essential in order to understand its reaction mechanism. In order to analyze the structure and function of KAS III, the fabH (Xoo4209) gene was cloned and the enzyme was expressed and purified. The KASIII crystal diffracted to 2.05 A resolution and belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 69.8, b = 79.5, c = 62.3 A. The unit-cell volume of the crystal is compatible with the presence of a single monomer in the asymmetric unit, with a corresponding Matthews coefficient V(M) of 2.27 A(3) Da(-1) and a solvent content of 45.8%.

The gltX gene from Xanthomonas oryzae pv. oryzae (Xoo1504) encodes glutamyl-tRNA synthetase (GluRS), one of the most important enzymes involved in bacterial blight (BB), which causes huge production losses of rice worldwide. GluRS is a class I-type aminoacyl-tRNA synthetase (aaRS) that is primarily responsible for the glutamylation of tRNA(Glu). It plays an essential role in protein synthesis, as well as the regulation of cells, in all organisms. As it represents an important target for the development of new antibacterial drugs against BB, determination of the three-dimensional structure of GluRS is essential in order to understand its catalytic mechanism. In order to analyze its structure and function, the gltX gene was cloned and the GluRS enzyme was expressed, purified and then crystallized. A GluRS crystal belonging to the monoclinic space group C2 diffracted to 2.8 A resolution and had unit-cell parameters a = 186.8, b = 108.4, c = 166.1 A, beta = 96.3 degrees . The unit-cell volume of the crystal allowed the presence of six to eight monomers in the asymmetric unit, with a corresponding Matthews coefficient (V(M)) range of 2.70-2.02 A(3) Da(-1) and a solvent-content range of 54.5-39.3%.

Nudix hydrolases are a family of proteins that contains the Nudix signature of the characteristic amino-acid sequence Gx(5)Ex(5) [UA]xREx(2)EExGU, where x represents any amino acid and U usually a bulky hydrophobic amino acid, such as Leu, Val, or Ile. They ubiquitously exist in more than 200 species. YmfB, a novel Nudix hydrolase found in Escherichia coli, is a nonspecific nucleoside tri- and di-phosphatase, which atypically releases inorganic orthophosphate from triphosphates instead of pyrophosphate. In this study, YmfB was cloned, overexpressed, and crystallized. Two different crystals, one belonging to an orthorhombic space group C222(1) and the other a monoclinic space group P2(1), diffracted to 2.7 A and 2.6 A resolution, and had unit cell parameters of a = 68.7 A, b = 283.1 A, c = 70.4 A and a = 69.1 A, b = 70.3 A, c = 145.6 A, beta = 103.3 degrees , respectively. For the C222(1) space group, four or five monomers exist in the asymmetric unit, with a corresponding V(m) of 2.48 or 1.99 A(3) Da(-1) and a solvent content of 50.5 or 38.2%. For the P2(1) space group, eight or nine monomers exist in the asymmetric unit, with a corresponding V(m) of 2.49 or 2.21 A(3) Da(-1) and a solvent content of 50.7 or 44.5%.

Human ribosomal protein S3 (hRpS3) is a small ribosomal subunit showing apurinic/apyrimidinic (AP) lyase activity and has been suggested to play a role in the cellular DNA-damage response pathway. However, the functional interactions between hRpS3 and other base excision repair (BER) DNA glycosylases have not been reported. We identified, for the first time, the interaction between hRpS3 and human uracil-DNA glycosylase (hUNG) and investigated the functional consequences of this interaction. hRpS3 was shown to interact with hUNG in co-immunoprecipitation assay using transiently transfected HEK293 cells and GST pull-down assay using microbial expression systems. In an assay using a 5'-end-radiolabeled 39-mer oligonucleotide duplex containing a U/G mismatch, hRpS3 dramatically stimulated the uracil-excision activity of hUNG, whereas hRpS3 alone had no cleavage activity. Pre-incubation of hRpS3 with the U/G mismatch containing DNA duplex also increased the hUNG uracil-excision activity; however, hRpS3 did not increase the DNA binding activity of hUNG in a trapping assay of hUNG and the U/G mismatch containing DNA duplex using UV cross-linking. hRpS3 has been suggested to stimulate the uracil-excision activity of hUNG by enhancing its dissociation from AP sites and increasing its turn-over rate. The disruption of hRpS3 by small-interfering RNA (siRNA-hRpS3) transfection reduced the uracil-excision activity preserved in cell extracts, whereas the supplement of purified hRpS3 retained uracil-excision activity. These results strongly suggest that hRpS3 may be involved in the uracil-excision pathway, probably by participating in the DNA repair mechanism to remove uracil generated by the deamination of cytosine in DNA, and by preventing C/G-->T/A transition mutations.