In silico analysis of carbohydrate-binding pockets in the lectin genes from various species of Canavalia

Legumes are endowed with an opulent class of proteins called lectins that can detect tenuous variations in carbohydrate structures and bind them reversibly with high affinity and specificity. The genus Canavalia, in the family of Leguminosae, is considered to be an affluent source of lectin. An effort has been made to analyse the sequences encoded by the lectin gene and its carbohydrate binding pockets from three species of Canavalia, including C. virosa, C. rosea, and C. pubescens. Crude seed extract showed highest haemagglutination titer against buffalo RBCs and has high affinity to mannose and trehalose. Amplification of the lectin gene by gene-specific primers showed the presence of an 870 bp amplicon. Physicochemical characterization using various bioinformatic tools showed that the isoelectric point was below 7, suggesting that lectin molecules were acidic. A high aliphatic index and high instability index were observed, which indicated that lectin molecules were stable towards a wide range of temperatures. The occurrence of N-glycosylation sites at two sites was also identified in all three species. Prediction of secondary structure showed that approximately 59.05 %, 56.76 % and 54.88 % of the elements were random coils in the case of C. virosa, C. pubescens and C. rosea, respectively. Comparative modelling of the proteins and docking of hypothetical models with sugar moieties that inhibited the agglutination activity suggested that asparagine, serine, alanine, valine, tyrosine and threonine were the major residues involved in hydrogen bonding and other stacking interactions. This can further provide insights on its prospective antibiosis property.     

In silico structural and functional characterization and phylogenetic study of alkaline phosphatase in bacterium,Rhizobium leguminosarum (Frank 1879)

Phosphorus is one of the primary macronutrient of plants, which is present in soil. It is essential for normal growth and development of plants. Plants use inorganic form of phosphate but organic form can also be assimilated with the help of soil inhabiting bacteria. Alkaline phosphatase is an enzyme present in Rizobium bacteria. This enzyme is responsible for solubilization and mineralization of organic phosphate and makes it readily available for plants. In the present study, nine different strains of Rhizobium leguminosarum were selected for a detailed computational structural and functional characterization and phylogenetic studies of alkaline phosphatase. Amino acid sequences were retrieved from UniProt and saved in FASTA format for use in analysis. Phylogenetic analysis of these strains was done by using MEGA7. 3D structure prediction was performed by using online server I-Tasser. Galaxy Web and 3D Refine were used for structure refinement. The refined structures were evaluated using two validation servers, QMEAN and SAVES. Protein-protein interaction analysis was done by using STRING. For detailed functional characterization, Cofactor, Coach, RaptorX, PSORT and MEME were used. Overall quality of predicted protein models was above 80%. Refined and validated models were submitted into PMDB. Seven out of nine strains were closely related and other two were distantly related. Protein-Protein interaction showed no significant co-expression among the interaction partners.     

Properties of a new alkaline proteinase from Aspergillus niger

A. niger LCF 9 synthesizes a new aspergillopeptidase of potential interest in therapeutics. The properties and operating range of the enzyme were determined. It is a semi-alkaline aspergillopeptidase (EC 3.4.23.4) with one endopeptidase activity. Its pI is 4.10, its molecular weight is 21000 Da and its A1%(1 cm) at 280 nm is 9.75. It rapidly hydrolyzes casein and hemoglobin. Its optimal pH is 7.8 and optimal temperature is 45 degrees C. It is thermally labile above 40 degrees C but can be stabilized by adding calcium ions. It is inhibited by phenylmethylsulfonyl fluoride (PMSF), by ethylenediaminetetraacetic acid (EDTA) and by certain metals ions, e.g. copper, manganese and cobalt ions. It has no dipeptidase or tripeptidase activity and its esterase activity is weak. It has a high collagenase activity and is to our knowledge the only aspergillopeptidase that is active towards benzoyl-arginine p-nitroanilide (BAPNA).     

In silico fight against novel coronavirus by finding chromone derivatives as inhibitor of coronavirus main proteases enzyme

Structural Chemistry - Novel coronavirus, 2019-nCoV is a danger to the world and is spreading rapidly. Very little structural information about 2019-nCoV make this situation more difficult for drug...     

In silico identification of conserved microRNAs and their target transcripts from expressed sequence tags of three earthworm species

MicroRNAs are a recently identified class of small regulatory RNAs that target more than 30% protein-coding genes. Elevating evidence shows that miRNAs play a critical role in many biological processes, including developmental timing, tissue differentiation, and response to chemical exposure. In this study, we applied a computational approach to analyze expressed sequence tags, and identified 32 miRNAs belonging to 22 miRNA families, in three earthworm species Eisenia fetida, Eisenia andrei, and Lumbricus rubellus. These newly identified earthworm miRNAs possess a difference of 2-4 nucleotides from their homologous counterparts in Caenorhabditis elegans. They also share similar features with other known animal miRNAs, for instance, the nucleotide U being dominant in both mature and pre-miRNA sequences, particularly in the first position of mature miRNA sequences at the 5' end. The newly identified earthworm miRNAs putatively regulate mRNA genes that are involved in many important biological processes and pathways related to development, growth, locomotion, and reproduction as well as response to stresses, particularly oxidative stress. Future efforts will focus on experimental validation of their presence and target mRNA genes to further elucidate their biological functions in earthworms.     

Thiol-dependent serine alkaline proteases from Bacillus sp. HR-08 and KR-8102

Two Bacillus sp. strains, HR-08 and KR-8102, isolated from soil of the west and north parts of Iran were screened on gelatin agar medium for their ability to produce alkaline protease. The enzymes were active in a wide pH range (6.0–11.0) and stable in the alkaline range (7.0–12.0). The optimum temperatures for the protease from HR-08 and KR-8102 were 65 and 50°C, respectively. The irreversible thermoinactivation of HR-08 and KR-8102 proteases showed that the stability of HR-08 enzyme was higher than that of KR-8102 and the half-lives of these enzymes were 95 and 32 min at 50°C, respectively. In the presence of 10 mM Ca²⁺, HR-08 retained 100, 90, and 20% of its initial activity after heating for 30 min at 50, 60, and 70°C, respectively. Enzymes were inhibited by phenylmethylsulfonyl fluoride and iodoacetate. After inhibition by iodoacetate, both enzymes were reactivated by dithiothreitol. These data show that the enzymes seem to be thiol-dependent serine alkaline proteases. The enzymes especially from HR-08 were stable in the presence of H₂O₂, surfactants, and local detergents; their activities were enhanced in the presence of 5 mM Fe²⁺; and the presence of 5mM metal ions such as Mg²⁺, Cu²⁺, and Mn²⁺ produced almost no effect.     

In silico prediction of drug toxicity

It is essential, in order to minimise expensive drug failures due to toxicity being found in late development or even in clinical trials, to determine potential toxicity problems as early as possible. In view of the large libraries of compounds now being handled by combinatorial chemistry and high-throughput screening, identification of putative toxicity is advisable even before synthesis. Thus the use of predictive toxicology is called for. A number of in silico approaches to toxicity prediction are discussed. Quantitative structure-activity relationships (QSARs), relating mostly to specific chemical classes, have long been used for this purpose, and exist for a wide range of toxicity endpoints. However, QSARs also exist for the prediction of toxicity of very diverse libraries, although often such QSARs are of the classification type; that is, they predict simply whether or not a compound is toxic, and do not give an indication of the level of toxicity. Examples are given of all of these. A number of expert systems are available for toxicity prediction, most of them covering a range of toxicity endpoints. Those discussed include TOPKAT, CASE, DEREK, HazardExpert, OncoLogic and COMPACT. Comparative tests of the ability of these systems to predict carcinogenicity show that improvement is still needed. The consensus approach is recommended, whereby the results from several prediction systems are pooled. It is simply amazing that we can formulate any kind of QSAR. The (desired activity) is only the starting point. The truly formidable problem is that of toxicity, especially the difficult long-term toxicities resulting from chronic usage'. (Hansch & Leo [1])     

In silico discovery of beta-secretase inhibitors

Alzheimer's disease, the most common amyloid-associated disorder, accounts for the majority of the dementia diagnosed after the age of 60. The cleavage of the beta-amyloid precursor protein is initiated by beta-secretase (BACE-1), a membrane-bound aspartic protease, which has emerged as an important but difficult protein target. Here, an in silico screening approach consisting of fragment-based docking, ligand conformational search by a genetic algorithm, and evaluation of free energy of binding was used to identify low-molecular-weight inhibitors of BACE-1. More than 300,000 small molecules were docked and about 15,000 prioritized according to a linear interaction energy model with evaluation of solvation by continuum electrostatics. Eighty-eight compounds were tested in vitro, and 10 of them showed an IC(50) value lower than 100 microM in a BACE-1 enzymatic assay. Interestingly, the 10 active compounds shared a triazine scaffold. Moreover, four of them were active in an assay with mammalian cells (EC(50) < 20 microM), indicating that they are cell-permeable. Therefore, these triazine derivatives are very promising lead candidates for BACE-1 inhibition. The discoveries of this series and two other series of nonpeptidic BACE-1 inhibitors demonstrate the usefulness of our in silico high-throughput screening approach.     

Purification and characterization of an exoinulinase from Aspergillus fumigatus

An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa, thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing, with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively. The inulin hydrolysis activity was completely abolished with 1 mM Hg⁺⁺, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower K _m (0.25 mM) and higher V _max (333.3 IU/mg) values for inulin.     

In silico modelling of ADMET-a minireview of work from 2000 to 2004

This article represents a minireview of work published so far in the 21st century in the in silico ADMET field of research related to investigations in the areas of solubility, hERG and cytochrome P450 3A4. Various approaches including 2D- and 3D-QSARs and pharmacophore modelling are discussed. The pros and cons of the methods used and models derived are examined. More general remarks on model development and validation are also reported.     

In silico identification of the protein disulfide isomerase family from a protozoan parasite

Protein disulfide isomerase (PDI) enzymes are eukaryotic oxidoreductases that catalyze the correct formation of disulfide bonds during protein folding. Structurally they are characterized by the presence of functional thioredoxin-like (Trx) domains. For the protozoan parasite causative of the human amebiasis (Entamoeba histolytica), the correct formation of disulfide bonds is important for an accurate folding of its proteins, including some virulence factors. However, little is known about the enzymes involved in this mechanism. We undertook a post-genomic approach to identify the PDI family of this parasite. The genome database survey revealed a set of 11 PDI-encoding sequences with predictable protein thiol/disulfide oxidoreductase activities.     

In silico prediction of deleterious single amino acid polymorphisms from amino acid sequence

Molecular cause of human disease retains as one of the most attractive scientific research targets for decades. An effective approach toward this topic is analysis and identification of disease-related amino acid polymorphisms. In this work, we developed a concise and promising deleterious amino acid polymorphism identification method SeqSubPred based on 44 features solely extracted from protein sequence. SeqSubPred achieved surprisingly good predictive ability with accuracy (0.88) and area under receiver operating characteristic (0.94) without resorting to homology or evolution information, which is frequently used in similar methods and usually more complex and time-consuming. SeqSubPred also identified several critical sequence features obtained from random forests model, and these features brought some interesting insights into the factors affecting human disease-related amino acid substitutions. The online version of SeqSubPred method is available at montana.informatics.indiana.edu/cgi-bin/seqmut/seqsubpred.cgi     

环境致癌物的计算机预测研究

环境致癌物可诱发人类或哺乳动物体内的肿瘤,建立环境致癌物的计算机预测模型对环境风险评价和生态安全具有重要的意义．通过构建了3780个化合物的数据集,随机选取其中3024个作为训练集,其余756个作为外部验证集;基于定量构-效关系（QSAR）方法,采用逐步判别分析和主成分分析建立数学模型．结果表明训练集非致癌物预测正确率为86．0％,可能致癌物的预测正确率为88．O％,而采用主成分建模时,非致癌物和可能致癌物的预测正确率分别为74．2％和73．1％．说明逐步判别分析法的结果优于主成分判别分析．同时确定了可能致癌物和非致癌物的分子结构参数,阐明了两者结构差异．以上结果为预测和评估环境致癌物提供参考依据．     

In silico Complexes of Amino Acids and Diamondoids

We report on the specific interaction of a small diamond-like molecule, known as diamondoid, with single amino-acids forming nano/bio molecular complexes. Using time-dependent density-functional theory calculations we have studied two different relative configurations of three prototypical amino acids, phenylalanine, tyrosine, and tryptophan, with the diamondoid. The optical and charge-transfer properties of these complexes exhibit amino acid and topology specific features which can be directly utilized for in the direction of novel biomolecule detection schemes.     

来源于类芽孢杆菌属碱性甲壳素酶的分离纯化及其性质

甲壳素,又名几丁质(chitin),是自然界中含量仅次于纤维素的第二大天然多糖,有第六生命要素之美称.其主要存在于甲壳类动物的外壳、真菌细胞的细胞壁以及一些昆虫的外壳中,每年自然界中约有100多亿吨甲壳素生成.甲壳素是由2-乙酰氨基-2-脱氧-D-吡喃葡萄糖和2-氨基-2-脱氧-D-吡喃葡萄糖通过β-1,4糖苷键连接而成的二元线性聚合物,分子链中分布许多羟基、氨基及乙酰氨基,形成大量分子间及分子内氢键,致使其结晶度较高,化学性质十分稳定,直接利用较为困难.甲壳素不溶于稀酸、稀碱以及一般有机溶剂,工业上常用强酸强碱法处理甲壳素,以制备壳寡糖类产品,但该方法具有产品结构不单一,环境污染较为严重等缺点.甲壳素酶可特异性水解甲壳素链中β-1,4糖苷键,得到甲壳寡糖和N-乙酰氨基葡萄糖.酶解法降解甲壳素工艺简单、反应条件温和、环境友好,有很好的应用前景.我们以Paenibacillus pasadenensis CS0611为出发菌株,以蟹壳粉末为培养基唯一碳源及氮源,在适宜条件下培养48 h.发酵液经离心、硫酸铵(80％饱和度)盐析、透析除盐后得到粗酶液.再利用HiTrap DEAE FF离子交换层析和HiLoad 26/600Superdex 200 pg凝胶过滤层析对该粗酶液进行分离纯化,以得到电泳纯甲壳素酶.所制备甲壳素酶比活力为10.28 U/mg,最终纯化倍数为5.3,酶活得率为15.7％.SDS-PAGE结果表明,该甲壳素酶相对分子质量约为69 kDa.后经MALDI-TOF-MS鉴定,该酶部分肽段和来源于另一株Paenibacillus pasadenenss的甲壳素酶(accession No:gi655151624)具有较高的同源性,进一步证实所纯化蛋白为甲壳素酶.对上述纯化的甲壳素酶的酶学性质进行研究,结果发现:其最适反应温度为50℃,在20-35℃内有较好的稳定性,50℃及以上热稳定性较差;最适pH为5.0,在pH4.0-11.0间具有较高稳定性,表明该酶具有很好的耐碱性;金属离子对该酶催化活性没有明显的激活作用,表明该甲壳素酶是非金属酶.同时,对该酶的底物特异性进行研究,发现该酶对胶体甲壳素和甲壳素水解能力较强,对淀粉和纤维素无水解能力,对不同脱乙酰度的壳聚糖的水解程度随脱乙酰度不同而变化,表明该酶只能特异性识别并降解GlcNAc-GlcNAc之间的糖苷键;以胶体甲壳素为底物时,米氏常数Km为4.41 mg/mL,最大反应初速度为1.08 mg/min.利用薄板层析和高效液相色谱对酶解产物进行分析,结果表明该甲壳素酶对胶体甲壳素的降解产物主要是(GlcNAc)2.综上所述,本研究所涉甲壳素酶在甲壳二糖的酶法制备方面具有较好的应用前景.