ExPASy Server Research Articles

Abstract 6916: InnoPanelTMand InnoPediaTM: resources of engineered cell lines and xenografts for drug discovery

Abstract The Development of precision medicine relies on well-characterized experimental models that carry naive, induced, or engineered genomic and epigenetic features targetable by certain treatments. Patient-derived xenografts (PDX) and cell lines can serve as such in vivo and in vitro models by harboring abnormalities such as somatic and driver mutations, dysregulated gene expression and silencing, microsatellite instability, gene fusions, copy number amplification and loss of heterozygosity. In addition, cell lines can also be engineered to introduce sometimes rare-occurring abnormalities that the engineered cell line shares an identical genetic background with its parental cell line, thus providing a clearly contrasting pair to evaluate treatment specificity. With the advancement of disease research and drug discovery, existing cell lines can no longer meet the needs of various types of drug discovery. To evaluate drug efficacy more quickly and accurately, Kyinno has constructed and identified more than 4,000 engineered cell lines, including overexpressed cell lines constructed through transposon systems or lentivirus infection, gene-edited cell lines constructed through CRISPR-Cas9 technology, and reporter cell lines targeting multiple signaling pathways and membrane receptors. Besides, InnoPanelTM, a panel of over 1000 engineered cell lines was established and characterized to cover a wide range of heavily targeted genes such as CLDN18.1, CLDN18.2, LAG3, OX40, PD-L1, PD-L2, and TIM3. Cell line families were also developed for genes with multiple known driver mutations. All cell lines are annotated with background information, engineering details, functional characterization, and efficacy studies. Furthermore, we developed xenografts for several engineered cell lines and showed that the combination of cell lines and xenografts can accelerate drug development. Additionally, all of this information can be accessed in our free database, InnoPediaTM. The database also offers browsing, searching, and analytical capabilities for over 600 commonly used tumor cell lines and thousands of engineered cell lines with essential genomic and pharmacological data, cell line efficacy data for nearly 2000 common anti-tumor drugs, and comprehensive details for over 100 standard antibodies. Our engineered cell lines are now registered in the online cell line database Cellosaurus, which is available on the ExPASy server. Citation Format: Siyu Li, Chang Liu, Na Wang, Yuqing Hu, Jinying Ning, Feng Hao. InnoPanelTMand InnoPediaTM: resources of engineered cell lines and xenografts for drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6916.

In-silico selection of appropriate signal peptides for romiplostim secretory production in Escherichia coli

BackgroundThrombocytopenia is a prevalent condition caused by a reduction in platelet production, increased platelet depletion, or a combination of both processes. Romiplostim, the most potent drug and the only thrombopoietin (TPO) receptor agonist, consists of four TPO receptor–activating peptides fused as a fusion protein to the human IgG1 heavy chain segment. Romiplostim could be produced by recombinant DNA technology in Escherichia coli. Extracellular and periplasmic secretion using signal peptides (SPs) of the recombinant proteins could have several advantages in purification and protein recovery. SPs are short amino-acid sequences used in protein translocation through various membranes. The structure of the different SPs identified in various bacteria and their compatibility with the protein could affect the transfer success and efficacy. ObjectiveThis study aimed to identify the best SP for extracellular secretion of romiplostim to facilitate its purification and reduce production costs in E. coli. MethodsThe amino acid sequences of the SPs and the position of their cleavage sites in the proteins were evaluated using the Signal 5.0 server. The solubility of the proteins produced in E. coli was predicted by protein-sol. The physicochemical parameters of the SPs were analyzed by ProtParam using the ExPASy server. Results/ConclusionWe found that SfmC is the most suitable candidate, followed by TorT and bla. However, this result should be confirmed by further experimental assessment. Furthermore, for the successful secretion of a recombinant protein, the optimal growth conditions should be controlled in addition to a suitable SP.

Homology Modeling Odorant-binding Protein-1 (OBP1) Anopheles Farauti Protein Target for Mosquito Repellent

The odorant binding protein (OBP) has a role as a target protein for potential interaction mechanism activity for the development of repellent compounds. The purpose of this study was to analyze the physico-chemical properties of the protein, the stability of the three-dimensional structure of the OBP1 Anopheles farauti protein, and to predict the binding site pocket as the target of the active protein site against inhibitors. Analysis of physico-chemical properties was carried out by the ProtParam Expasy server. The theoretical calculated isoelectric point (pI) was found to be less than 7 indicating the acidic nature of this protein. The aliphatic index of 78 indicates the thermal stability of the protein. The Grand Average of Hydropathicity (GRAVY) is estimated at -0.355; This lower GRAVY value indicates a possible better interaction of this protein with water. Secondary structure analysis was carried out by SOPMA which revealed that Alpha helix (55.86%) predominated among the secondary structural elements followed by Random coil (32.41%), Extended strand (8.97%), and Beta turn (2.76%). Three-dimensional structure modeling of OBP1 Anopheles farauti was performed with the Swiss-Model server and the protein refine Galaxy server. Homology modeling results obtained PDB ID 2ERB template with sequence identity 94.4%. The model was validated for the three-dimensional structure of the protein using the MolProbity, ProSA, ProQ, ERRAT, Verfy3D, and PROCHECK servers. The prediction results of pocket binding sites using DoSiteScore obtained three pocket binding site locations, namely P_0 (Drug score 0.84); P_1 ((0.75); P_2 (Drug score 0.64). Conclusion Homology modeling of the protein OBP1 Anopheles farauti has been carried out and the three-dimensional structure of the Model_OBP1_04 protein has been obtained that meets the criteria for valid structural parameters so that this structure can be used In-silico molecular docking and molecular dynamic studies for the development of mosquitoes repellent.

In silico structural, phylogenetic and drug target analysis of putrescine monooxygenase from Shewanella putrefaciens-95

BackgroundThe enormous and irresponsible use of antibiotics has led to the emergence of resistant strains of bacteria globally. A new approach to combat this crisis has been nutritional immunity limiting the availability of nutrients to pathogens. Targeting the siderophore biosynthetic pathway that helps in iron acquisition, an essential microelement in the bacterial system has been the topic of interest in recent days that backs the concept of nutritional immunity. Supporting this view, we have chosen to study a key enzyme in the biosynthetic pathway of putrebactin called putrescine monooxygenase (SpPMO) from Shewanella putrefaciens. In our previous study, we co-expressed putrescine monooxygenase recombinantly in Escherichia coli BL21 Star (DE3). The bioinformatic analysis and screening of inhibitors will broaden the scope of SpPMO as a drug target. ResultsIn the present study, we have analysed the physicochemical properties of the target enzyme and other N-hydroxylating monooxygenases (NMOs) using ExPASy server. The target enzyme SpPMO and most of the selected NMOs have a slightly acidic isoelectric point and are medially thermostable and generally insoluble. The multiple sequence alignment identified the GXGXX(N/A), DXXXFATGYXXXXP motives and conserved amino acids involved in FAD binding, NADP binding, secondary structure formation and substrate binding. The phylogenetic analysis indicated the distribution of the monooxygenases into different clades according to their substrate specificity. Further, a 3D model of SpPMO was predicted using I-TASSER online tool with DfoA from Erwinia amylovora as a template. The model was validated using the SAVES server and deposited to the Protein Model Database with the accession number PM0082222. The molecular docking analysis with different substrates revealed the presence of a putrescine binding pocket made of conserved amino acids and another binding pocket present on the surface of the protein wherein all other ligands interact with high binding affinity. The molecular docking of naturally occurring inhibitor molecules with SpPMO 3D model identified curcumin and niazirin with 1.83 and 2.81 μM inhibition constants as two promising inhibitors. Further studies on kinetic parameters of curcumin and niazirin inhibitors in vitro determined the Ki to be 2.6±0.0036 μM and 18.38±0.008 μM respectively. ConclusionThis analysis will help us understand the structural, phylogenetic and drug target aspects of putrescine monooxygenase from Shewanella putrefaciens-95 in detail. It sheds light on the precautionary measures that can be developed to inhibit the enzyme and thereby the secondary infections caused by them. Graphical abstract▪ Highlights•The physicochemical properties of putrescine monooxygenase from Shewanella putrefaciens-95 were compared with other known N-hydroxylating monooxygenases.•The phylogenetic analysis of SpPMO and other selected class B monooxygenases clearly classified NMOs according to their substrate affinity.•The molecular docking analysis of various substrates to SpPMO enzyme showed the presence of two binding pockets.•Curcumin and niazirin were identified as two potential natural inhibitors to SpPMO enzyme with 2.6±0.0036 μM and 18.38±0.008 μM inhibition constants respectively.

In-silico evaluation of the potential of a viral protein (apoptin) as an anticancer agent

As a significant immunosuppressive virus, chicken anemia virus (CAV) is common among natural poultry hosts and newborn chickens. It contains a tiny single-stranded negative circular DNA genome, encoding three proteins vp1, vp2, and vp3. Apoptin, encoded by the vp3 gene of the CAV virus, is a small protein whose subcellular localization appears to be crucial for tumor-selective activity. Residing in the cytoplasm of normal cells, it translocates into the nucleus in cancerous cells. Apoptin has attracted much attention because of causes specific death in transformed and cancer cells and has toxic effects on cancer cells. In-silico analysis of apoptin revealed the unknown structural and functional features. Physicochemical features and sequence analysis and secondary structure prediction were also conducted using Expasy server tools. Furthermore, the global structure of apoptin was modeled using the I-TASSER server and antigenic features were obtained via the application of the IEBD web tools. The results of in-silico analysis indicated that this protein had a stable structure, was a suitable choice for therapeutic goals, capable of acting as a good anti-cancer agent.

Structure and function prediction analysis of Leptin

Objective Leptin is an important protein hormone produced by white adipose tissue, which participates in the regulation of energy metabolism. Its research on improving metabolic mechanism after bariatric surgery is attracting more and more attention. The purpose of this study is to analyze Leptin’s structure and predict its function by bioinformatics, in order to further explore the role of leptin in the treatment of obesity by bariatric surgery. Methods Tools of ExPASy Server were employed to analyze the physicochemical properties of Leptin protein. The secondary structure was predicted by Jpred 4 and its three-level structure by Swiss-Mode. STRINGS database is used to predict interactions between Leptin and other proteins. Results The Leptin protein consisted of 167 amino acids with an isoelectric point of 5.88, an instability index of 47.52, and a fat coefficient of 110.84. There were 5 α-helicesand 1 β-sheet in the secondary structure prediction. The number of amino acids constituting alpha helix accounted for 50.9% of the total protein. Three possible tertiary structure models were simulated. From the prediction model, the protein was mainly alpha-helix, which was consistent with the prediction results of secondary structure. According to the trend of similar waveform, prediction model A is the most stable. The protein interaction network found that the protein families interacting with Leptin include Leptin receptor (LEPR), Signal transducer and activator of transcription 3 (STAT3), Peroxisome proliferator-activated receptor gamma (PPARG), Suppressor of cytokine signaling 3 (SOCS3), Glucagon (GCG), Tyrosine-protein kinase JAK2, Insulin receptor substrate 1 (IRS1), C-reactive protein (CRP), Tyrosine-protein phosphatase non-receptor type 1 (PTPN1) and Insulin (INS). Conclusion Leptin protein is an unstable lipophilic protein mainly composed of alpha-helix. It is secreted by white adipose tissue and mainly acts on the outside of cells. It inhibits the synthesis of adipocytes by participating in the regulation of sugar, fat and energy metabolism, thus reducing body weight. Key words: Leptin; Bariatric Surgery; Bioinformatics

CLASTR: The Cellosaurus STR similarity search tool - A precious help for cell line authentication.

Despite an increased awareness of the problematic of cell line cross-contamination and misidentification, it remains nowadays a major source of erroneous experimental results in biomedical research. To prevent it, researchers are expected to frequently test the authenticity of the cell lines they are working on. STR profiling was selected as the international reference method to perform cell line authentication. While the experimental protocols and manipulations for generating a STR profile are well described, the available tools and workflows to analyze such data are lacking. The Cellosaurus knowledge resource aimed to improve the situation by compiling all the publicly available STR profiles from the literature and other databases. As a result, it grew to become the largest database in terms of human STR profiles, with 6,474 distinct cell lines having an associated STR profile (release July 31, 2019). Here we present CLASTR, the Cellosaurus STR similarity search tool enabling users to compare one or more STR profiles with those available in the Cellosaurus cell line knowledge resource. It aims to help researchers in the process of cell line authentication by providing numerous functionalities. The tool is publicly accessible on the SIB ExPASy server (https://web.expasy.org/cellosaurus-str-search) and its source code is available on GitHub under the GPL-3.0 license.

The ABCD database: a repository for chemically defined antibodies

The ABCD (for AntiBodies Chemically Defined) database is a repository of sequenced antibodies, integrating curated information about the antibody and its antigen with cross-links to standardized databases of chemical and protein entities. It is freely available to the academic community, accessible through the ExPASy server (https://web.expasy.org/abcd/). The ABCD database aims at helping to improve reproducibility in academic research by providing a unique, unambiguous identifier associated to each antibody sequence. It also allows to determine rapidly if a sequenced antibody is available for a given antigen.

Comparison between Echinococcus granulosus sensu stricto (G1) and E. canadensis (G6) mitochondrial genes (cox1 and nad1) and their related protein models using experimental and bioinformatics analysis

BackgroundCystic echinococcosis (CE) as a zoonotic parasitic disease, remains a health challenge in many parts of the world. There are different species of Echinococcus granulosus sensu lato with different pathogenicity and host preferences.Different procedures have been applied for characterization of Echinococcus taxa in which two mitochondrial genes, cox1 and nad1 have been used more common. They have been able to differentiate E. granulosus sensu stricto and E. canadensis species in different hosts. The affinity of E. granulosus sensu stricto and E. canadensis species for localizing different organs seems to be different. To what such affinity and related pathogenicity could be related, is not known, so far. Bioinformatics analysis may be helpful to interpret such difference by investigating the genes and their related protein models between different species infecting human and animals. The current work was designed to study the differences between E. granulosus s.s. and E. canadensis species mitochondrial genes (cox1 and nad1) and related protein models of CE cysts by experimental and bioinformatics analysis. Materials and methodsDifferent human and animal CE cysts were collected and their DNA was extracted and sequenced based on their cox1 and nad1 genes. In order to determine the E. granulosus s.s. and E. canadensis species of the samples, BLAST analysis was performed on sequenced genes. Three sequences were selected for analysis and were deposited in GenBank. Moreover, the sequence number of KT988116.1 which belonged to E. canadensis from our already deposited in GenBank was also selected. Alignment and phylogenetic analysis were performed on the sequences using BioEdit and MEGA7 software. The raw sequences of translated proteins belonged to the mentioned genes were obtained from Protein database in NCBI. The secondary structure was determined by PSIPRED Protein Sequence Analysis Workbench. The tertiary models of COX1 and NAD1 proteins in both genotypes were constructed using Modeler 9.12 software and their physicochemical features were computed using ProtParam tool in ExPASY server. ResultsBLAST analysis on sequenced genes showed that the samples belonged to E. granulosus s.s. and E. canadensis species. These sequences were deposited in GenBank with accession numbers: JN579173.1, KF437811.1, and KY924632.1.The results showed that proteins of COX1 of E. granulosus s.s., COX1of E. canadensis, NAD1of E. granulosus s.s. and NAD1of E. canadensis species, consisted of 135, 122, 120 and 124 amino acids, respectively. The aligned sequences of translated proteins belonged to COX1 and NAD1 enzymes in E. granulosus s.s. and E. canadensis species were different; such that alignment COX1 sequence between E. granulosus s.s. and E. canadensis species showed that amino acids were different in 6 positions. This difference for NAD1 sequences were different in 19 positions. The secondary structure determined by PSIPRED showed differences in coil, strand and helix chains in COX1 and NAD1 proteins in E. granulosus s.s. and E. canadensis species. Comparison between three-dimensional structures (3D) of COX1 protein model in E. granulosus s.s. and E. canadensis species demonstrated an additional helix with two conserved iron binding sites in the COX1 protein of E. granulosus s.s. species. ConclusionE. granulosus s.s. and E. canadensis species differences are reflected in two important proteins: COX1 and NAD1. These differences are demonstrable in the 3D structure of proteins of both strains. So, the present study is adding to our understanding of the difference in molecular sequences between the E. granulosus s.s. (G1) and E. canadensis (G6) which may be used for interpreting the difference between the pathogenicity and localization affinity in these two important helminthic zoonosis.

The Cellosaurus, a Cell-Line Knowledge Resource.

The Cellosaurus is a knowledge resource on cell lines. It aims to describe all cell lines used in biomedical research. Its scope encompasses both vertebrates and invertebrates. Currently, information for >100,000 cell lines is provided. For each cell line, it provides a wealth of information, cross-references, and literature citations. The Cellosaurus is available on the ExPASy server (https://web.expasy.org/cellosaurus/) and can be downloaded in a variety of formats. Among its many uses, the Cellosaurus is a key resource to help researchers identify potentially contaminated/misidentified cell lines, thus contributing to improving the quality of research in the life sciences.

Identification and Physiochemical Analysis of ERK Interacting Proteins Using Bio-Computational Tools

ERK (Extracellular Signal Regulated Kinase) or MAP kinase is an intracellular signaling molecule. ERK is involved in regulation of various functions i.e. cell proliferation, cell migrations, cell survival and many more. It gets activated in response of various stimuli like growth factors, cytokines, virus, second messengers, transforming agents and carcinogens. While transferring signals from cell surface receptors to cell nucleus, ERK interacts with a numbers of proteins. Physiochemical and functional characterization of these proteins is little known. Thus, we attempted to study physiochemical and functional properties of ERK interacting proteins using bio-computational tools. ExPASy and SOSUI server suggested 22 ERK interacting proteins. Physical and chemical parameters of these ERK interacting partners indicated higher percentage of hydrophobic amino acid and leucine as major constituent. Moreover, the instability index indicated that four proteins are stable in over wide range temperature in vitro, and remaining eighteen proteins were found unstable. In addition, SOSUI server showed that fifteen proteins were soluble and six are trans-membrane in nature.

Genome-wide bioinformatics analysis of Dof transcription factor gene family of chickpea and its comparative phylogenetic assessment with Arabidopsis and rice

The genome mining of chickpea (Cicer arietinum L.) revealed a total of 37 putative Dof genes using NCBI BLAST search against the genome with a highly conserved Dof domain. The translated Dof proteins possessed 150–493 amino acid residues with molecular weight ranging from 16.9 to 54.4 kD and pI varied from 4.98 to 9.64 as revealed by ExPASy server ProtParam. The exon–intron organization showed predominance of intronless Dof genes in chickpea. The predicted Dof genes were distributed among the eight chromosomes with a maximum of 9 Dof genes present on chromosome 7 and a single Dof gene was found on chromosome 8.The predominance of segmental gene duplication as compared to tandem duplication was observed which might be the prime cause of Dof gene family expansion in chickpea. The cis-regulatory element analysis revealed the presence of light-responsive, hormone-responsive, endosperm-specific, meristem-specific and stress-responsive elements. Comprehensive phylogenetic analyses of Dof genes of chickpea with Arabidopsis, rice, soybean and pigeonpea revealed several orthologs and paralogs assisting in understanding the putative functions of CaDof genes. The functional divergence and site-specific selective pressures of chickpea Dof genes have been investigated. The bioinformatics-based genome-wide assessment of Dof gene family of chickpea attempted in the present study could be a significant step for deciphering novel Dof genes based on genome-wide expression profiling.

In silico Designing of Protein Rich in Large Neutral Amino Acids Using Bovine αs1 Casein for Treatment of Phenylketonuria

Phenylketonuria (PKU) is a genetically inherited disease where the body fails to convert Phenylalanine (Phe) to Tyrosine (Tyr) due to the defective Phenylalanine Hydroxylase (PAH) enzyme, resulting in the elevated blood Phe level causing neurological damage. Of all therapies, Large Neutral Amino Acid (LNAA) supplementation has become a promising approach to the dietary treatment of PKU, where the LNAA compete with Phe for the same L-Type Large Neutral Amino Acid Transporter (LAT1, SLC7A5) across the blood-brain barrier, decreasing brain Phe level. In this study we have designed an easily digestible protein enriched with LNAA (except Phe), using bovine αs1 casein as template by homology modeling. Our challenge was to maximize the LNAAs (except Phe) in the protein model by finding a suitable scaffold for hosting LNAAs, thereby turning the usual concept of homology modeling. Bioinformatics tools like SWISS-MODEL, EXPASY, PROFUNC, I-TASSER, RaptorX, and SAVeS Server were used for the structure prediction, and validation of the designed protein. Out of 63 different models designed, Protein Model-54 was selected based on sequence similarity to template (61.4%), compact 3D structure containing only α-helices and coils without β-sheets (QMEAN score of 0.567), and good in silico digestibility. The molecular weight of protein was 12,094.6 Da. Ramachandran plot revealed that the designed protein contained 89.9% amino acid residues in the favored region with ERRAT score of 88.04. Based on these evaluations, the Protein Model-54 was found to be the best, stable and reliable model which may be of high nutritional significance for PKU patients..

In-silico Analysis and Homology Modeling of Cytochrome C Oxidase Sub Unit I from Indian Major Carp, <i>Labeo rohita</i>

Labeo rohita is the popular table fish in the whole Indian sub-continent. Cytochrome c oxidase sub unit I (COX/COI) is the largest protein coding gene in the metazoan mitochondrial genome and COI is the most frequently studied region of the teleost mitochondrial genome. It is the terminal member of the respiratory chain catalyzing the reduction of dioxygen to water by ferrocytochrome C. In this study, COI of L. rohita (56.86KDa) has been characterized, and the 3- D structure was predicted by using Modeller version 9.12 by homology modeling. The secondary structure has been predicted using PSIPRED and TMHMM server, and the predicted model has been validated in RAMPAGE server. The protein statistics was carried out by using Protopram tool in Expasy server. The predicted 3-D model showed that 99.8% of residues have Φ and Ψ angle in the favored and allowed regions. It was found to be a transmembrane protein and consisted of an N-terminal signal peptide. The isoelectric focusing point, instability index, aliphatic index, grand average hydropathicity, extinction coefficient, absorbance and number of negatively charged and positively charged amino acids were; 6.05, 26.52, 109.81, 0.749, 121180, 2.142, 25 and 16 respectively, revealing the structure and properties of COI protein. The COX/CO-I protein sequences of Catla catla, Cirrhinus mrigala, Labeo rohita, C. cirrhosus and Danio rerio were also taken for the divergence study and mutation analysis.

Two structurally different dienelactone hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 plasmid pJP4 catalyse cis- and trans-dienelactones with similar efficiency.

In this study, dienelactone hydrolases (TfdEI and TfdEII) located on plasmid pJP4 of Cupriavidus necator JMP134 were cloned, purified, characterized and three dimensional structures were predicted. tfdEI and tfdEII genes were cloned into pET21b vector and expressed in E. coli BL21(DE3). The enzymes were purified by applying ultra-membrane filtration, anion-exchange QFF and gel-filtration columns. The enzyme activity was determined by using cis-dienelactone. The three-dimensional structure of enzymes was predicted using SWISS-MODEL workspace and the biophysical properties were determined on ExPASy server. Both TfdEI and TfdEII (Mr 25 kDa) exhibited optimum activity at 37°C and pH 7.0. The enzymes retained approximately 50% of their activity after 1 h of incubation at 50°C and showed high stability against denaturing agents. The TfdEI and TfdEII hydrolysed cis-dienelactone at a rate of 0.258 and 0.182 µMs−1, with a Km value of 87 µM and 305 µM, respectively. Also, TfdEI and TfdEII hydrolysed trans-dienelactone at a rate of 0.053 µMs−1 and 0.0766 µMs−1, with a Km value of 84 µM and 178 µM, respectively. The TfdEI and TfdEII kcat/Km ratios were 0.12 µM−1s−1and 0.13 µM−1s−1 and 0.216 µM−1s−1 and 0.094 µM−1s−1 for for cis- and trans-dienelactone, respectively. The kcat/Km ratios for cis-dienelactone show that both enzymes catalyse the reaction with same efficiency even though Km value differs significantly. This is the first report to characterize and compare reaction kinetics of purified TfdEI and TfdEII from Cupriavidus necator JMP134 and may be helpful for further exploration of their catalytic mechanisms.

Cloning, expression, purification and three-dimensional structure prediction of haloalkane dehalogenase from a recently isolated Ancylobacter aquaticus strain UV5

Haloalkane dehalogenase (DhlA) converts 1,2-dichloroethane (1,2-DCA) to 2-chloroethane in the genus Ancylobacter and Xanthobacter autotrophicus GJ10 (XaDhlA) and allows these organisms to utilise 1,2-DCA and some other halogenated alkanes for growth. The DhlA encoding gene (dhlA) was PCR-amplified from the genomic DNA of a recently isolated Ancylobacter aquaticus UV5 strain, cloned and overexpressed in Escherichiacoli BL21 (DE3). The recombinant enzyme was purified by using Amicon ultra-15 centrifugal filter units, an anion-exchange QFF column followed by a gel-filtration column (Sephacryl HR100). Enzyme activity was determined by using 1,2-DCA as a substrate. Three-dimensional structure of the enzyme was predicted using SWISS-MODEL workspace and the biophysical properties were predicted by submitting the amino acid sequence of DhlA on ExPASy server. DhlA (Mr 35kDa) exhibited optimum activity at temperature 37°C and pH 9.0. The enzyme retained approximately 50% of its activity after 1h of incubation at 50°C, and showed moderate stability against denaturing agent urea. The DhlA displayed a Km value of 842μM and kcat/Km ratio of 168mM−1min−1 for its substrate 1,2-DCA. This DhlA was found to belong to the α/β hydrolase family with a catalytic triad composed of Asp-His-Asp in its active site. This is the first study reporting on the characterisation and reaction kinetics of purified DhlA from A.aquaticus UV5 indigenous to contaminated site in Africa.

Inherent Regulation of EAL Domain-catalyzed Hydrolysis of Second Messenger Cyclic di-GMP

The universal second messenger cyclic di-GMP (cdG) is involved in the regulation of a diverse range of cellular processes in bacteria. The intracellular concentration of the dinucleotide is determined by the opposing actions of diguanylate cyclases and cdG-specific phosphodiesterases (PDEs). Whereas most PDEs have accessory domains that are involved in the regulation of their activity, the regulatory mechanism of this class of enzymes has remained unclear. Here, we use biophysical and functional analyses to show that the isolated EAL domain of a PDE from Escherichia coli (YahA) is in a fast thermodynamic monomer-dimer equilibrium, and that the domain is active only in its dimeric state. Furthermore, our data indicate thermodynamic coupling between substrate binding and EAL dimerization with the dimerization affinity being increased about 100-fold upon substrate binding. Crystal structures of the YahA-EAL domain determined under various conditions (apo, Mg(2+), cdG·Ca(2+) complex) confirm structural coupling between the dimer interface and the catalytic center. The built-in regulatory properties of the EAL domain probably facilitate its modular, functional combination with the diverse repertoire of accessory domains.

Mass Isotopomer Analysis of Metabolically Labeled Nucleotide Sugars and N- and O-Glycans for Tracing Nucleotide Sugar Metabolisms

Nucleotide sugars are the donor substrates of various glycosyltransferases, and an important building block in N- and O-glycan biosynthesis. Their intercellular concentrations are regulated by cellular metabolic states including diseases such as cancer and diabetes. To investigate the fate of UDP-GlcNAc, we developed a tracing method for UDP-GlcNAc synthesis and use, and GlcNAc utilization using (13)C6-glucose and (13)C2-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS. Metabolic labeling of cultured cells with (13)C6-glucose and the analysis of isotopomers of UDP-HexNAc (UDP-GlcNAc plus UDP-GalNAc) and CMP-NeuAc revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and use. In pancreatic insulinoma cells, the labeling efficiency of a (13)C6-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells. Using (13)C2-glucosamine, the diversity of the labeling efficiency was observed in each sugar residue of N- and O-glycans on the basis of isotopomer analysis. In the insulinoma cells, the low labeling efficiencies were found for sialic acids as well as tri- and tetra-sialo N-glycans, whereas asialo N-glycans were found to be abundant. Essentially no significant difference in secreted hyaluronic acids was found among hepatoma and insulinoma cell lines. This indicates that metabolic flows are responsible for the low sialylation in the insulinoma cells. Our strategy should be useful for systematically tracing each stage of cellular GlcNAc metabolism.

SugarBind database (SugarBindDB): a resource of pathogen lectins and corresponding glycan targets

SugarBindDB lists pathogen and biotoxin lectins and their carbohydrate ligands in a searchable format. Information is collected from articles published in peer-reviewed scientific journals. Help files guide the user through the search process and provide a review of structures and names of sugars that appear in human oligosaccharides. Glycans are written in the condensed form of the carbohydrate nomenclature system developed by the International Union of Pure and Applied Chemistry (IUPAC). Since its online publication by The MITRE Corporation in 2005, the database has served as a resource for research on the glycobiology of infectious disease. SugarBindDB is currently hosted by the Swiss Institute of Bioinformatics on the ExPASy server and will be enhanced and linked to related resources as part of the wider UniCarbKB initiative. Enhancements will include the option to display glycans in a variety of formats, including modified 2D condensed IUPAC and symbolic nomenclature.

An Extra Peptide within the Catalytic Module of a β-Agarase Affects the Agarose Degradation Pattern

Agarase hydrolyzes agarose into a series of oligosaccharides with repeating disaccharide units. The glycoside hydrolase (GH) module of agarase is known to be responsible for its catalytic activity. However, variations in the composition of the GH module and its effects on enzymatic functions have been minimally elucidated. The agaG4 gene, cloned from the genome of the agarolytic Flammeovirga strain MY04, encodes a 503-amino acid protein, AgaG4. Compared with elucidated agarases, AgaG4 contains an extra peptide (Asn(246)-Gly(302)) within its GH module. Heterologously expressed AgaG4 (recombinant AgaG4; rAgaG4) was determined to be an endo-type β-agarase. The protein degraded agarose into neoagarotetraose and neoagarohexaose at a final molar ratio of 1.5:1. Neoagarooctaose was the smallest substrate for rAgaG4, whereas neoagarotetraose was the minimal degradation product. Removing the extra fragment from the GH module led to the inability of the mutant (rAgaG4-T57) to degrade neoagarooctaose, and the final degradation products of agarose by the truncated protein were neoagarotetraose, neoagarohexaose, and neoagarooctaose at a final molar ratio of 2.7:2.8:1. The optimal temperature for agarose degradation also decreased to 40 °C for this mutant. Bioinformatic analysis suggested that tyrosine 276 within the extra fragment was a candidate active site residue for the enzymatic activity. Site-swapping experiments of Tyr(276) to 19 various other amino acids demonstrated that the characteristics of this residue were crucial for the AgaG4 degradation of agarose and the cleavage pattern of substrate.