野葛葡糖基转移酶PlUGTs的同源建模及其活性位点分析

本文对PIUGTs进行同源建模,并分析其与底物结合的构象及活性位点。通过SWISS-MODEL在线对P1UGTs进行模板预测和选择,运用Swiss—PdbViewer软件显示和优化,利用ACDLABS绘制糖基供体小分子（酶结合底物）,最后通过AutoDock_ADT进行分子对接,并分析PIUGTs酶与不同底物结合的整体构象及分析活性位点。研究结果表明PIUGT1、PIUGT2及PIUGT3均能得到较好的三级构象,并且PIUGT1、PIUGT2与三种底物均可进行较好对接,H18,R278,N359为PIUGTI与三种对接构象活性中心所共有的氨基残基;而G16,H17,V19,T148,N370,E374,E390为PIUGT2与三种对接构象活性中心所共有的氨基残基,但PIUGT3未能得到较好的对接构象。由此推测PIUGT1和PIUGT2均能合成葛根素．而PIUGT3不能催化葛根素的合成。     

Homology modeling of Ferredoxin-nitrite reductase from Arabidopsis thaliana

Different subtypes of Influenza A virus are associated with species specific, zoonotic or pandemic Influenza. The cause of its severity underlies in complicated evolution of its segmented RNA genome. Although genetic shift and genetic drift are well known in the evolution of this virus, we reported the significant role of unique RNA palindromes in its evolution. Our computational approach identified the existence of unique palindromes in each subtype of Influenza A virus with its absence in Influenza B relating the fact of virulence and vigorous genetic hitchhiking in Influenza A. The current study focused on the re-assortment event responsible for the emergence of pandemic-2009 H1N1 virus, which is associated with outgrow of new palindrome and in turn, changing its RNA structure. We hypothesize that the change in RNA structure due to the presence of palindrome facilitates the event of re-assortment in Influenza A. Thus the evolutionary process of Influenza A is much more complicated as previously known, and that has been demonstrated in this study.     

Homology Modeling and Comparative Profiling of Superoxide Dismutase Among Extremophiles: Exiguobacterium as a Model Organism

Superoxide dismutase (SOD), a well known antioxidant enzyme, is known to exert its presence across bacteria to humans. Apart from their well-known antioxidant defense mechanisms, their association with various extremophiles in response to various stress conditions is poorly understood. Here, we have discussed the conservation and the prevalence of SODs among 21 representative extremophiles. A systematic investigation of aligned amino acid sequences of SOD from all the selected extremophiles revealed a consensus motif D-[VLE]-[FW]-E-H-[AS]-Y-[YM]. To computationally predict the correlation of SOD with the various stress conditions encountered by these extremophiles, Exiguobacterium was selected as a model organism which is known to survive under various adverse extremophilic conditions. Interestingly, our phylogenetic study based on SOD homology revealed that Exiguobacterium sibiricum was one of the closest neighbors of Deinococcus radiodurans and Thermus thermophilus. Next, we sought to predict 3-D model structure of SOD for E. sibiricum (PMDB ID: 0078260), which showed >95 % similarity with D. radiodurans R1 SOD. The reliability of the predicted SOD model was checked by using various validation metrics, including Ramachandran plot, Z-score and normalized qualitative model energy analysis score. Further, various physicochemical properties of E. sibiricum SOD were calculated using different prominent resources.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-014-0482-8) contains supplementary material, which is available to authorized users.     

Isolation and in-silico characterization of Peroxidase isoenzymes from Wheat (Triticum aestivum) against Karnal Bunt (Tilletia indica)

To investigate the role of Peroxidase and its physiological significance under Karnal Bunt (KB) were determined in resistant (HD-29) and susceptible genotype (WH-542) of wheat during different developmental stages. The enzymes were expressed constitutively in both the susceptible and resistant genotype. In gel assay and differential expression analysis of POD was significantly higher (p >0.05) in Sv and S2, than the S1 and S3 stages. in silico analysis of Peroxidase for eg. physico-chemical properties, secondary structural features and phylogenetic classification for comparative analysis. Motif and Domain analysis of Peroxidase by MEME, to be important for the biological functions, and studies of evolution. Our results clearly indicate that the enhanced expression of POD at the WS2 stage, which reinforces its role in stage dependent immunity against Karnal bunt and role of POD metabolism provides genotype and stage dependant structural barrier resistance in wheat against KB.     

Expression, Purification and Characterization of the Proline Dehydrogenase Domain of PutA from Pseudomonas putida POS-F84

The objective of the present work was to express a truncated form of Pseudomonas putida PutA that shows proline dehydrogenase (ProDH) activity. The putA gene encoding ProDH enzyme was cloned into pET23a vector and expressed in Escherichia coli strain BL-21 (DE3) plysS. The recombinant P. putida enzyme was biochemically characterized and its three dimensional structure was also predicted. ProDH encoding sequence showed an open reading frame of 1,035-bp encoding a 345 amino acid residues polypeptide chain. Purified His-tagged enzyme gave a single band with a molecular mass of 40 kDa on SDS-PAGE. The molecular mass of the isolated enzyme was found to be about 40 kDa by gel filtration. This suggested that the enzyme of interest consists of one subunit. The K _m and V _max values of recombinant P. putida ProDH were estimated to be 31 mM and 132 μmol/min, respectively. The optimum pH and temperature for the catalytic activity of the enzyme was about pH 8.5 and 30 °C. The modeling analysis of the three dimensional structure elucidated that Ser-165, Lys-195 and Ala-252 were key residues for the ProDH activity. This study provides data on the cloning, sequencing and recombinant expression of PutA ProDH domain from P. putida POS-F84.     

Myostatin三维结构模建及分子进化分析

Myostatin(MST)为肌肉生长负调节因子,其功能受抑制可导致肌肉量增加.对MST核酸序列进行序列比对,构建进化树;采用同源模建方法首次模建MST成熟肽生物活性二聚体的四级结构,并预测MST与其受体ActRIIB的相互作用模式.进化树将肌肉生长抑制素基因(MSTN)分成4个亚家族:哺乳动物MSTN,鸟类MSTN以及鱼类MSTN 1和2.MST受纯化选择作用,在不同物种的直系同源基因具有较高的刚源性,其中哺乳动物、鸟类MST C端活性肽氨基酸序列高度保守.表明哺乳动物、鸟类MST的结构、功能类似,且信号传导路径可能一致;而鱼类MST的调控机制可能存在较大差异.MST结构及其表面静电势和疏水氨基酸分布表明静电力和疏水相互作用在MST与其受体结合过程中可能起到十分重要的作用.     

铁皮石斛甘露糖结合凝聚素的分子建模与对接研究

本文通过序列分析获得了铁皮石斛甘露糖结合凝聚素(Dendrobium officinale mannose-binding lectin,DOL)成熟肽和甘露糖结合位点(50-58AA,81-89AA,116-124AA)。通过同源建模建立了DOL三维结构模型,DOL呈中空的三棱柱结构,三棱柱的三个侧面主要由β折叠构成,三个侧面各有一个甘露糖结合部位。甘露糖与DOL的分子对接和动力学分析表明,结合位点50-58AA和81-89AA对甘露糖的结合要强于116-124AA,在与甘露糖结合的过程中发挥关键作用的氨基酸残基为Gln81,Asp83,Asn85和Tyr89。研究结果有助于进一步开展凝集素抗病机理及凝集素相关药物研究。     

Structural characterization of a flavonoid glycosyltransferase from Withania somnifera

Medicinal plants are extensively utilized in traditional and herbal medicines, both in India and around the world due to the presence of diverse low molecular weight natural products such as flavonoids, alkaloids, terpenoids and sterols. Flavonoids which have health benefits for humans are the large class of phenylpropanoid-derived secondary metabolites and are mostly glycosylated by UDP-glycosyltransferases (UGTs). Although large numbers of different UGTs are known from higher plants, very few protein structures have been reported till now. In the present study, the three-dimensional model of flavonoid specific glycosyltransferases (WsFGT) from Withania somnifera was constructed based on the crystal structure of plant UGTs. The resulted model was assessed by various tools and the final refined model revealed GT-B type fold. Further, to understand the sugar donors and acceptors interactions with the active site of WsFGT, docking studies were performed. The amino acids from conserved PSPG box were interacted with sugar donor while His18, Asp110, Trp352 and Asn353 were important for catalytic function. This structural and docking information will be useful to understand the glycosylation mechanism of flavonoid glucosides.

Abbreviations

DOPE - Discrete Optimized Potential Energy, PDB - Protein Data Bank, PSPG - Plant Secondary Product Glycosyltransferase, RMSD - Root Mean Squared Deviation, UDP - Uridine diphosphate, UGT - UDP-glycosyltransferases.     

人脑和人血清胆碱酯酶三维结构的计算机模拟研究

本文以同源的电鳐胆碱酯酶（Ｔ．ＡＣｈＥ）的三维结构为模板,模拟预测了人脑和血清胆碱酯酶（Ｈ。ＡＣｈＥ和Ｈ．ＢｕＣｈＥ）的三维结构和活力中心的组成。指Ｔ．ＡＣｈＥ,Ｈ．ＡＣｈＥ和Ｈ．ＢｕＣｈＥ宁间结构差异,并讨论了ＡＣｈ和Ｈ。ＡＣｈＥ的对接（ｄｏｃｋｉｎｇ）。Ｈ。ＡＣｈＥ和Ｈ．ＢｕＣｈＥ三维结构的确定将为进一步深入研究它的中毒机理和合理药物设计提供靶子。     

产甘油假丝酵母与酿酒酵母胞浆3-磷酸甘油脱氢酶基因的功能比较

胞浆3-磷酸甘油脱氢酶(GPD)是酿酒酵母细胞甘油合成过程中的关键限速酶．尽管高产甘油菌株产甘油假丝酵母基因组中编码该酶的基因CgGPD已经被克隆出来,但是具体的功能,特别是与酿酒酵母GPD1和GPD2基因的功能比较值得进一步研究．以酿酒酵母渗透压敏感型的gpd1/gpd2和gpd1突变株为宿主,分别导入CgGPD、GPD1和GPD2基因,比较分析了CgGPD、GPD1和GPD2基因在高渗透压胁迫条件下和厌氧环境中的表达调控,及其对细胞甘油合成能力的影响．研究发现,GPD1基因受到渗透压诱导表达,GPD2基因在细胞厌氧条件下起着氧化还原平衡调节作用,而CgGPD基因不仅能够在渗透压胁迫条件下通过过量快速合成甘油调节渗透压平衡,而且能够在厌氧培养环境中互补GPD2基因的缺失,使gpd1/gpd2缺失突变株能够正常生长,同时提高了突变株的甘油合成能力．结果表明,CgGPD基因在gpd1/gpd2缺失突变株中既具有GPD1基因的功能,又能发挥GPD2基因的功能．     

Homology Modeling of Coagulase in Staphylococcus aureus

The close correlation between the ability of coagulase to clot blood plasma and their capacity to produce disease, and the corresponding absence of this property in nonpathogenic strains, have led to the assumption that the coagulase, plays important role in the pathogenesis of disease. Currently, crystal structure of coagulase in Staphylococcus aureus remains indefinable. Thus, the objectives of this research is to generate the three dimensional model of coagulase in S. aureus by using homology modeling approach. In this study, we used bioinformatics tools and databases such as BLAST (Basic Local Alignment Search Tool), GenBank, PDB (Protein Databank), and Discovery Studio to gain specific functional insights into coagulase. The model was validated using protein structure checking tools such as PROCHECK, Verify 3D and CE (Combinatorial Extension) for reliability. Therefore, structure prediction of coagulase in S. aureus can provide preliminary knowledge for understanding the function of the protein. The information from this finding will provide important information into the action and regulation mechanism of the coagulase protein in S. aureus.     

Metabolic pathway analysis and molecular docking analysis for identification of putative drug targets in Toxoplasma gondii: novel approach

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that can infect a wide range of warm-blooded animals including humans. In humans and other intermediate hosts, toxoplasma develops into chronic infection that cannot be eliminated by host's immune response or by currently used drugs. In most cases, chronic infections are largely asymptomatic unless the host becomes immune compromised. Thus, toxoplasma is a global health problem and the situation has become more precarious due to the advent of HIV infections and poor toleration of drugs used to treat toxoplasma infection, having severe side effects and also resistance have been developed to the current generation of drugs. The emergence of these drug resistant varieties of T. gondii has led to a search for novel drug targets. We have performed a comparative analysis of metabolic pathways of the host Homo sapiens and the pathogen T. gondii. The enzymes in the unique pathways of T. gondii, which do not show similarity to any protein from the host, represent attractive potential drug targets. We have listed out 11 such potential drug targets which are playing some important work in more than one pathway. Out of these, one important target is Glutamate dehydrogenase enzyme; it plays crucial part in oxidation reduction, metabolic process and amino acid metabolic process. As this is also present in the targets of tropical diseases of TDR (Tropical disease related Drug) target database and no PDB and MODBASE 3D structural model is available, homology models for Glutamate dehydrogenase enzyme were generated using MODELLER9v6. The model was further explored for the molecular dynamics simulation study with GROMACS, virtual screening and docking studies with suitable inhibitors against the NCI diversity subset molecules from ZINC database, by using AutoDock-Vina. The best ten docking solutions were selected (ZINC01690699, ZINC17465979, ZINC17465983, ZINC18141294_03, ZINC05462670, ZINC01572309, ZINC18055497_01, ZINC18141294, ZINC05462674 and ZINC13152284_01). Further the Complexes were analyzed through LIGPLOT. On the basis of Complex scoring and binding ability it is deciphered that these NCI diversity set II compounds, specifically ZINC01690699 (as it has minimum energy score and one of the highest number of interactions with the active site residue), could be promising inhibitors for T. gondii using Glutamate dehydrogenase as Drug target.     

Kinetic and Structural Characterization for Cofactor Preference of Succinic Semialdehyde Dehydrogenase from Streptococcus pyogenes

The γ-Aminobutyric acid (GABA) that is found in prokaryotic and eukaryotic organisms has been used in various ways as a signaling molecule or a significant component generating metabolic energy under conditions of nutrient limitation or stress, through GABA catabolism. Succinic semialdehyde dehydrogenase (SSADH) catalyzes the oxidation of succinic semialdehyde to succinic acid in the final step of GABA catabolism. Here, we report the catalytic properties and two crystal structures of SSADH from Streptococcus pyogenes (SpSSADH) regarding its cofactor preference. Kinetic analysis showed that SpSSADH prefers NADP⁺ over NAD⁺ as a hydride acceptor. Moreover, the structures of SpSSADH were determined in an apo-form and in a binary complex with NADP⁺ at 1.6 Å and 2.1 Å resolutions, respectively. Both structures of SpSSADH showed dimeric conformation, containing a single cysteine residue in the catalytic loop of each subunit. Further structural analysis and sequence comparison of SpSSADH with other SSADHs revealed that Ser158 and Tyr188 in SpSSADH participate in the stabilization of the 2’-phosphate group of adenine-side ribose in NADP⁺. Our results provide structural insights into the cofactor preference of SpSSADH as the gram-positive bacterial SSADH.     

Homology modeling, molecular docking and electrostatic potential analysis of MurF ligase from Klebsiella pneumonia

In spite of availability of moderately protective vaccine and antibiotics, new antibacterial agents are urgently needed to decrease the global incidence of Klebsiella pneumonia infections. MurF ligase, a key enzyme, which participates in the bacterial cell wall assembly, is indispensable to existence of K. pneumonia. MurF ligase lack mammalian vis-à-vis and have high specificity, uniqueness, and occurrence only in eubacteria, epitomizing them as promising therapeutic targets for intervention. In this study, we present a unified approach involving homology modeling and molecular docking studies on MurF ligase enzyme. As part of this study, a homology model of K. pneumonia (MurF ligase) enzyme was predicted for the first time in order to carry out structurebased drug design. The accuracy of the model was further validated using different computational approaches. The comparative molecular docking study on this enzyme was undertaken using different phyto-ligands from Desmodium sp. and a known antibiotic Ciprofloxacin. The docking analysis indicated the importance of hotspots (HIS 281 and ASN 282) within the MurF binding pocket. The Lipinski's rule of five was analyzed for all ligands considered for this study by calculating the ADME/Tox, drug likeliness using Qikprop simulation. Only ten ligands were found to comply with the Lipinski rule of five. Based on the molecular docking results and Lipinki values 6-Methyltetrapterol A was confirmed as a promising lead compound. The present study should therefore play a guiding role in the experimental design and development of 6-Methyltetrapterol A as a bactericidal agent.     

Homology modeling and substrate binding study of Nudix hydrolase Ndx1 from Thermos thermophilus HB8

With homology modeling techniques, molecular mechanics, and molecular dynamics methods, a 3D structure model of Ndx1 is created and refined. This model is further assessed by Profile-3D and ProStat, which confirm that the refined model is reliable. With this model, a flexible docking study is performed and the result indicates that Glu46, Arg88, and Glu90 are three important determinant residues in binding, as they have strong hydrogen bonding interactions and electrostatic interactions with Ap6A. In addition, we further find that three residues, Ser38, Leu39 and Glu46, coordinate enzyme-bound Mg2+ ions in complex N-A. The Glu46 is consistent with the experimental results by Iwai et al., and the other four residues mentioned above may also play vital roles in catalysis of Ndx1.     

Structural and Functional Characterization of Plant Aminoaldehyde Dehydrogenase from Pisum sativum with a Broad Specificity for Natural and Synthetic Aminoaldehydes

Aminoaldehyde dehydrogenases (AMADHs, EC 1.2.1.19) belong to the large aldehyde dehydrogenase (ALDH) superfamily, namely, the ALDH9 family. They oxidize polyamine-derived ω-aminoaldehydes to the corresponding ω-amino acids. Here, we report the first X-ray structures of plant AMADHs: two isoenzymes, PsAMADH1 and PsAMADH2, from Pisum sativum in complex with β-nicotinamide adenine dinucleotide (NAD⁺) at 2.4 and 2.15 Å resolution, respectively. Both recombinant proteins are dimeric and, similarly to other ALDHs, each monomer is composed of an oligomerization domain, a coenzyme binding domain and a catalytic domain. Each subunit binds NAD⁺ as a coenzyme, contains a solvent-accessible C-terminal peroxisomal targeting signal (type 1) and a cation bound in the cavity close to the NAD⁺ binding site. While the NAD⁺ binding mode is classical for PsAMADH2, that for PsAMADH1 is unusual among ALDHs. A glycerol molecule occupies the substrate binding site and mimics a bound substrate. Structural analysis and substrate specificity study of both isoenzymes in combination with data published previously on other ALDH9 family members show that the established categorization of such enzymes into distinct groups based on substrate specificity is no more appropriate, because many of them seem capable of oxidizing a large spectrum of aminoaldehyde substrates. PsAMADH1 and PsAMADH2 can oxidize N,N,N-trimethyl-4-aminobutyraldehyde into γ-butyrobetaine, which is the carnitine precursor in animal cells. This activity highly suggests that in addition to their contribution to the formation of compatible osmolytes such as glycine betaine, β-alanine betaine and γ-aminobutyric acid, AMADHs might participate in carnitine biosynthesis in plants.     

Homology Modeling and Docking Studies of Human Bcl-2L10 Protein

Abstract

Bradykinin is a bioactive hormone involved in a variety of physiological processes. In various solvents, this peptide adopts β-turn structures. The C-terminal turn is a structural feature for the receptor affinity of agonists and antagonists while the N-terminal turn might be important for antagonistic activities. Polyphenols like dimeric proanthocyanidin B3 interact with the peptide. Thus to investigate the effects of polyphenols on bradykinin activity and structure, we studied the interaction in the structuring solvent DMSO which can be a close mimic of aqueous physiological environments like receptor-binding sites. Bradykinin alone presented a folded structure with two turns. B3 interacted with the peptide C-terminus and involved the loss of the bend structure of this region, while the N-ter-minus turn was maintained. Numerous studies have shown that polyphenolic molecules can act upon various biological targets, and the formation of this type of complex might be one of the possible modes of action.     

M1毒蕈碱乙酰胆碱受体同源建模模板的选取及验证

目的:探讨不同同源模板所获得M1毒蕈碱乙酰胆碱受体模型的合理性及可靠性。方法:以牛视紫红素受体、人源β2-肾上腺素受体、M2胆碱受体和M3胆碱受体为模板,分别对M1胆碱受体进行同源建模;采用分子对接获得各M1胆碱受体同源模板与配体的互作模式,并与已报道的M胆碱受体晶体结构进行静态比对,得到最佳M1胆碱受体同源模板;采用分子动力学模拟分析配体与关键残基距离的变化,对M1胆碱受体同源模板进行动态验证。结果:M2胆碱受体与M1胆碱受体的序列相似度较高,为67.9%;以Inactive M2胆碱受体为模板构建的M1胆碱受体(M1R_(inactive-M2R))与其他晶体结构间RMSD值的均值最低,为1.39;M1R_(inactive-M2R)别构位点K392及E397残基侧链与结合口袋距离更近,与配体结合构象更匹配;分子对接结果显示,双位点别构激动剂VU0184670与M1R_(inactive-M2R)别构结合位点Y85、Y381的距离分别为4.8、6.8,优于其他模型;分子动力学模拟后,配体与Q177残基的距离由7.4降至2.9,提示配体VU0184670向Q177方向偏转,与文献结果一致。结论:以Inactive M2受体结构为模板构建的M1胆碱受体模型最为合理,更接近M1胆碱受体的晶体结构。本研究为M1胆碱受体药物开发提供重要工具,为其他GPCRs受体同源建模提供创新范式。     

PCR-based molecular characterization and insilico analysis of food-borne trematode parasites Paragonimus westermani, Fasciolopsis buski and Fasciola gigantica from Northeast India using ITS2 rDNA

In early 2009, new swine-origin influenza A (H1N1) virus emerged in Mexico and the United States. The emerging influenza virus had made global influenza pandemic for nearly one year. To every emerging pathogen, exploring the origin sources is vital for viral control and clearance. Influenza virus is different from other virus in that it has 8 segments, making the segment reassortment a main drive in virus evolution. In exploring reassortment evolution origins of a newly emerging influenza virus, integrated comparing of the origin sources of all the segments is necessary. If some segments have high homologous with one parental strain, lower homologous with another parental strain, while other segments are reverse, can we proposed that this emerging influenza virus may re-assort from the two parental strains. Here we try to explore the multilevel reassortment evolution origins of 2009 H1N1 influenza virus using this method. By further validating the fidelity of this strategy, this method might be useful in judging the reassortment origins of newly emerging influenza virus.     

Homology modeling and PAPS ligand (cofactor) binding study of bovine phenol sulfotransferase

In order to understand the mechanisms of ligand binding and the interaction between the ligand and the bovine phenol sulfotransferase, (bSULT1A1, EC 2.8.2.1) a three-dimensional (3D) model of the bSULT1A1 is generated based on the crystal structure of the estrogen sulfotransferase (PDB code 1AQU) by using the InsightII/Homology module. With the aid of the molecular mechanics and molecular dynamics methods, the final refined model is obtained and is further assessed by Profile-3D and ProStat, which show that the refined model is reliable. With this model, a flexible docking study is performed and the results indicate that 3-phosphoadenosine-5- phosphosulfate (PAPS) is a more preferred ligand than coenzyme A (CoA), and that His108 forms hydrogen bond with PAPS, which is in good agreement with the experimental results. From these docking studies, we also suggest that Phe255, Phe24 and Tyr169 in bSULT1A1 are three important determinant residues in binding as they have strong van-der-Waals contacts with the ligand. The hydrogen–bonding interactions also play an important role for the stability of the complex. Our results may be helpful for further experimental investigations.Figure The final 3D-structure of bSULT1A1. The structure is obtained by energy minimizing an average conformation over the last 100 ps of MD simulation. The -helix is represented in red and the -sheet in yellow.