Characterized Till Date Research Articles

N-terminal PPE domain plays an integral role in extracellular transportation and stability of the immunomodulatory Rv3539 protein of the Mycobacterium tuberculosis

Multigene PE/PPE family is exclusively present in mycobacterium species. Only few selected genes of this family have been characterized till date. Rv3539 was annotated as PPE63 with conserved PPE domain at N-terminal and PE-PPE at C-terminal. An α/β hydrolase structural fold, characteristic of lipase/esterase, was present in the PE-PPE domain. To assign the biochemical function to Rv3539, the corresponding gene was cloned in pET-32a (+) as full-length, PPE, and PE-PPE domains individually, followed by expression in E. Coli C41 (DE3). All three proteins demonstrated esterase activity. However, the enzyme activity in the N-terminal PPE domain was very low. The enzyme activity of Rv3539 and PE-PPE proteins was approximately same with the pNP-C4 as optimum substrate at 40 °C and pH 8.0. The loss of enzyme activity after mutating the predicted catalytic triad (Ser296Ala, Asp369Ala, and His395Ala) found only in the PE-PPE domain, confirmed the candidature of the bioinformatically predicted active site residue. The optimal activity and thermostability of the Rv3539 protein was altered by removing the PPE domain. CD-spectroscopy analysis confirmed the role of PPE domain to the thermostability of Rv3539 by maintaining the structural integrity at higher temperatures. The presence of the N-terminal PPE domain directed the Rv3539 protein to the cell membrane/wall and the extracellular compartment. The Rv3539 protein could generate humoral response in TB patients. Therefore, results demonstrated that Rv3539 demonstrated esterase activity. PE-PPE domain of Rv3539 is functionally automated, however, N-terminus domain played a role in protein stabilization and its transportation. Both domains participated in immunomodulation.

Insilico analysis of peptides isolated from Agaricus bisporus manifests potential antimicrobial therapeutic activities

More and more microorganisms are progressively acquiring resistances to conventional antibiotics. Consequently, new antibiotics which are more effective are needed. Antimicrobial peptides (AMPs) are recognized as effective alternative to conventional antibiotics. The AMPs are obtained from different organisms including animals, plants, fungi, algae, and microorganisms. One of such significant sources of AMPs is Agaricus bisporus (button mushroom) that is long-familiar for its medicinal values. However, the occurrence and potential of AMPs in A. bisporus have not been well characterized till date. This study was aimed to identify AMPs within A. bisporus proteome and to further evaluate its antimicrobial potentials through in silico analysis. The proteome of A. bisporus was explored for antimicrobial peptides and their physicochemical properties were evaluated using bioinformatics tools. The proteome of A. bisporus contains 63 AMPs with ample antimicrobial properties such as broad spectrum efficacy, stable, non-allergenic and non-haemolytic attributes. It was further identified that these AMPs putatively target pathogens via membrane disruption and inhibition of ATP-dependent enzymes. This study renders a basis for further evaluation of identified AMPs through in vitro experimentations and trials to elucidate their practical use as therapeutic antimicrobial drugs. Resultantly, positive AMPs could be subjected to commercialization as cheaper and effective alternatives to conventional antibiotics

Pharmacological potential of ants and their symbionts – a review

AbstractAnts (Hymenoptera: Formicidae) play an important role in the pharmacological and food industry due to their therapeutic and nutritional properties. Being the most abundant eusocial insects in the world, ants also serve as a repository of bioactive compounds, which are utilized for their ecological interactions and defense. Ants have been used for the treatment of various diseases including asthma, cancer, arthritis, and other microbial infections in both modern and ethnic medicine. This is attributed to various compounds, such as antimicrobial peptides, biogenic amines, alkaloids, and flavonoids obtained from ant venom as well as whole‐body extracts. However, most of these compounds have not been characterized till date. Ant endosymbionts are also known to contribute to some of these properties and to play a crucial role in digestion, protection from enemies, nutritional upgrading, nitrogen recycling, and reproductive manipulation of their host. Hence, they have been used in pharmaceutical, food, and cosmetic industries due to their ability to produce various bioactive compounds. In contrast, ectosymbionts specifically help in pathogen defense. However, the potential of ant symbionts in the welfare and production of therapeutic compounds is not extensively studied. This review mainly focuses on the role of ants and their symbiotic microbes as a potential source of bioactive compounds in therapeutic interventions for exploring future possibilities in drug development research.

Molecular characterization, evolutionary and phylogenetic analyses of rice ACT/BAT-type amino acid transporters

The Amino Acid-Polyamine-Organocation (APC) family transporters containing BAT/ACT (amino acid/choline transporters), PHS/LAT (polyamine H+-symporters) and CAT (cationic amino acid transporters) type transporters are ubiquitously reported in plants. ACT (containing BAT genes) which mediates bidirectional amino acid transport has been poorly characterized till date. In the present study, bioinformatics-based analytical studies have been performed to characterize the structural, functional, and evolutionary features of seven ACT/BAT transporters in the model crop Oryza sativa. Sequence homologies with allied transporters suggest that these seven transporter proteins can also have important roles in regulating amino acid transportation. Evolutionary and phylogenetic study of OsBATs proteins using different statistical models based on CDS, Protein, and UTR regions have brought forward some valuable information regarding their way and patterns of diversification over the time, possibly due to the influence of their prevailing growth conditions. Computational analyses based on promoter regions, exon-intron structural patterns, conserved motifs, secondary and tertiary structures and ligand binding sites with probable ligand names provided clues on their evolutionary structural diversification indicating changes in their functional specification probably needed to combat with the changing environment.

Structural and functional characterization of a putative carbonic anhydrase from Geobacillus kaustophilus reveals its cambialistic function

Carbonic anhydrases (CA) are the most ubiquitous ancient zinc metalloenzymes known. Here we report the structural and functional analysis of a hypothetical protein GK2848 from Geobacillus kaustophilus. The analysis revealed that it belongs to the γ-class of CA (termed as Cag). Only a limited number of γ-class CA’s have been characterized till date. Interestingly Cag contains magnesium at its active site instead of a traditional zinc ion. Based on the structural and sequence comparison with similar γ-CA’s the putative active site residues of Cag were identified. This analysis revealed that an important catalytic residue and a proton shuttle residue (Glu62 and Glu84 respectively) of Cam (previously characterized γ-CA from Methanosarcina thermophila) are absent in Cag, however certain other active site residues are conserved both in Cag and Cam. This suggests that Cag uses a different set of residues for the reversible hydration of CO2 to HCO3− when compared with Cam. Inductively Coupled Plasma – Optical Emission Spectrometry (ICP-OES) and 25Mg and 67Zn NMR studies on Cag and its mutants revealed that either Mg or Zn can occupy the active site which suggests the cambialistic nature of the enzyme.

FtsA-FtsZ interaction in Vibrio cholerae causes conformational change of FtsA resulting in inhibition of ATP hydrolysis and polymerization

Proper interaction between the divisome proteins FtsA and FtsZ is important for the bacterial cell division which is not well characterized till date. In this study, the objective was to understand the mechanism of FtsA-FtsZ interaction using full-length recombinant proteins. We cloned, over-expressed, purified and subsequently characterized FtsA of Vibrio cholerae (VcFtsA). We found that VcFtsA polymerization assembly was dependent on Ca2+ ions, which is unique among FtsA proteins reported until now. VcFtsA also showed ATPase activity and its assembly was ATP dependent. Binding parameters of the interaction between the two full-length proteins, VcFtsA, and VcFtsZ determined by fluorescence spectrophotometry yielded a Kd value of around 38 μM. The Kd value of the interaction was 3 μM when VcFtsA was in ATP bound state. We found that VcFtsZ after interacting with VcFtsA causes a change of secondary structure in the later one leading to loss of its ability to hydrolyze ATP, subsequently halting the VcFtsA polymerization. On the other hand, a double-mutant of VcFtsA (VcFtsA-D242E,R300E), that does not bind to VcFtsZ, polymerized in the presence of VcFtsZ. Though FtsA proteins among different organisms show 70–80% homology in their sequences, assembly of VcFtsA showed a difference in its regulatory processes.

Correction: Structural insights into the nanomolar affinity of RING E3 ligase ZNRF1 for Ube2N and its functional implications.

RING (Really Interesting New Gene) domains in ubiquitin RING E3 ligases exclusively engage ubiquitin (Ub)-loaded E2s to facilitate ubiquitination of their substrates. Despite such specificity, all RINGs characterized till date bind unloaded E2s with dissociation constants (Kds) in the micromolar to the sub-millimolar range. Here, we show that the RING domain of E3 ligase ZNRF1, an essential E3 ligase implicated in diverse cellular pathways, binds Ube2N with a Kd of ∼50 nM. This high-affinity interaction is exclusive for Ube2N as ZNRF1 interacts with Ube2D2 with a Kd of ∼1 µM, alike few other E3s. The crystal structure of ZNRF1 C-terminal domain in complex with Ube2N coupled with mutational analyses reveals the molecular basis of this unusual affinity. We further demonstrate that the ubiquitination efficiency of ZNRF1 : E2 pairs correlates with their affinity. Intriguingly, as a consequence of its high E2 affinity, an excess of ZNRF1 inhibits Ube2N-mediated ubiquitination at concentrations ≥500 nM instead of showing enhanced ubiquitination. This suggests a novel mode of activity regulation of E3 ligases and emphasizes the importance of E3-E2 balance for the optimum activity. Based on our results, we propose that overexpression-based functional analyses on E3 ligases such as ZNRF1 must be approached with caution as enhanced cellular levels might result in aberrant modification activity.

Characterization of a novel amylosucrase gene from the metagenome of a thermal aquatic habitat, and its use in turanose production from sucrose biomass

Turanose is a natural isomer of sucrose. It is an emerging functional sweetener of the next generation. Turanose is catalytically synthesized from the sucrose biomass by employing amylosucrase enzyme. In this study, a novel gene encoding amylosucrase (Asmet) has been identified from the metagenome of a thermal aquatic habitat. Asmet exhibits 37–55% identity at the protein level with the known amylosucrases characterized till date. Asmet was cloned and expressed in Escherichia coli, followed by protein purification, and characterization. Asmet protein exhibited the maximum total activity at 9.0 pH and 60 °C temperature, whereas, 8.0 pH and 50 °C temperature were found optimum for transglycosylation activity. Asmet showed fairly high thermal tolerance at 50 °C. The conjugation of Asmet protein with functionalized iron nanoparticles significantly improved its thermal tolerance, showing hardly any loss in the enzyme’s activity even after 72 h of heat (50 °C) exposure. The turanose yield of about 47% was achieved from 1.5 M sucrose, containing 0.5 M fructose in the reaction. Turanose was purified (˜95%) via a bio-physical process, and characterized by TLC, HPLC, and NMR. The novel amylosucrase gene was demonstrated to be a potential candidate for turanose production, utilizing various sucrose containing feedstocks.

The four repeat Giardia lamblia telomere forms tetramolecular G-quadruplex with antiparallel topology

Guanine rich DNA sequences of regulatory genomic regions form secondary structures known as G-quadruplexes usually stabilized by tetrads of Hoogsteen hydrogen bonded guanines. The in vivo existence of G-quadruplexes ascertains their biological roles. Human telomeric repeats are the most studied G-rich sequences. The four repeat Giardia telomeric sequence (TAGGG)4 differs from its human counterpart (TTAGGG)4, by deletion of one T at the G-tract intervening site of each repeat. We show here that whilst the two repeat Giardia telomeric sequence (TAGGG)2 forms parallel and antiparallel quadruplexes with tetramolecular topology exclusively, the four repeat version (TAGGG)4 forms a tetramolecular (antiparallel) and unimolecular (parallel) quadruplexes in Na+. The tetramolecular (antiparallel) G-quadruplex formed by four repeats of Giardia telomeric sequence is stabilized by the additional Watson-Crick bonding between its intervening TA bases aligned in antiparallel fashion. Four stranded antiparallel quadruplex for four repeats of any telomeric sequence have not been characterized till date. We hypothesize that telomeric association in antiparallel fashion, (via G-overhangs to form tetramolecular quadruplex) could be a biologically relevant molecular event. Further, coexistence of Hoogsteen as well as Watson-Crick base pairing might give insight for recognition of conformationally diverse DNA structures by ligands. Communicated by Ramaswamy H. Sarma

Catalytic triad heterogeneity in S51 peptidase family: Structural basis for functional variability.

Peptidase E (PepE) is a nonclassical serine peptidase with a Ser-His-Glu catalytic triad. It is specific for dipeptides with an N-terminal aspartate residue (Asp-X dipeptidase activity). Its homolog from Listeria monocytogenes (PepElm) has a Ser-His-Asn "catalytic triad." Based on sequence alignment we predicted that the PepE homolog from Deinococcus radiodurans (PepEdr) would have a Ser-His-Asp "catalytic triad." We confirmed this by solving the crystal structure of PepEdr to 2.7 Å resolution. We show that PepElm and PepEdr lack the Asp-X dipeptidase activity. Our analyses suggest that absence of P1 pocket in the active site could be the main reason for this lack of typical activity. Sequence and structural data reveal that the PepE homologs can be divided into long and short PepEs based on presence or absence of a C-terminal tail which adopts a β-hairpin conformation in the canonical PepE from Salmonella enterica. A long PepE from Bacillus subtilis with Ser-His-Asp catalytic triad exhibits Asp-X dipeptidase activity. Whereas the three long PepEs enzymatically characterized till date have been found to possess the Asp-X dipeptidase activity, the three enzymatically characterized short PepEs lack this activity irrespective of the nature of their catalytic triads. This study illuminates the structural and functional heterogeneity in the S51 family and also provides structural basis for the functional variability among PepE homologs.

Prediction of microRNA and gene target from an integrated network in chronic obstructive pulmonary disease based on canonical correlation analysis.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a complex disorder with a high mortality. The pathophysiology of COPD has not been characterized till date.OBJECTIVE:To identify COPD-related biomarkers by a bioinformatics analysis.METHODS:Here, we conducted the canonical correlation analysis to extract the potential COPD-related miRNAs and mRNAs based on the miRNA-mRNA dual expression profiling data. After identifying miRNAs and mRNAs related to COPD, we constructed an interaction network by integrating three validated miRNA-target sources. Then we expanded the network by adding miRNA-mRNA pairs, which were identified by Spearman rank correlation test. For miRNAs involved in the network, we further performed the Gene Ontology (GO) functional enrichment analysis of their targets. To validate COPD-related mRNAs involved in the network, we performed receiver operating characteristic (ROC) curve analysis and Support Vector Machine (SVM) classification on only those mRNAs that overlapped with COPD-related mRNAs of Online Mendelian Inheritance in Man (OMIM) database. RESULTS:The results indicate that some identified miRNAs and their targets in the constructed network might be potential biomarkers of COPD.CONCLUSIONS: Our study helps us to predict the potential risk biomarkers of COPD, and it can certainly help in further elucidating the genetic etiology of COPD.

Structural insights into the nanomolar affinity of RING E3 ligase ZNRF1 for Ube2N and its functional implications

RING (Really Interesting New Gene) domains in ubiquitin RING E3 ligases exclusively engage ubiquitin (Ub)-loaded E2s to facilitate ubiquitination of their substrates. Despite such specificity, all RINGs characterized till date bind unloaded E2s with dissociation constants (Kds) in the micromolar to the sub-millimolar range. Here, we show that the RING domain of E3 ligase ZNRF1, an essential E3 ligase implicated in diverse cellular pathways, binds Ube2N with a Kd of ∼50 nM. This high-affinity interaction is exclusive for Ube2N as ZNRF1 interacts with Ube2D2 with a Kd of ∼1 µM, alike few other E3s. The crystal structure of ZNRF1 C-terminal domain in complex with Ube2N coupled with mutational analyses reveals the molecular basis of this unusual affinity. We further demonstrate that the ubiquitination efficiency of ZNRF1 : E2 pairs correlates with their affinity. Intriguingly, as a consequence of its high E2 affinity, an excess of ZNRF1 inhibits Ube2N-mediated ubiquitination at concentrations ≥500 nM instead of showing enhanced ubiquitination. This suggests a novel mode of activity regulation of E3 ligases and emphasizes the importance of E3-E2 balance for the optimum activity. Based on our results, we propose that overexpression-based functional analyses on E3 ligases such as ZNRF1 must be approached with caution as enhanced cellular levels might result in aberrant modification activity.

Discovery of novel highly active and stable aspartate dehydrogenases

Aspartate family amino acids (AFAAs) have important commercial values due to their wide spectrum of applications. Most if not all AFAAs are produced under aerobic conditions which is energy-intensive. To establish a cost-effective anaerobic process for production of AFAAs, it holds great promise to develop a new pathway enabling the conversion of oxoloacetate into aspartate through direct amination which is catalyzed by aspartate dehydrogenase (AspDH). Compared with the well studied aspartate aminotransferase and aspartate ammonia-lyase, only a few AspDHs are characterized till date, and failure to reproduce the high activity of AspDH from Rastonia eutropha documented in the literature encouraged us to screen and characterize novel AspDHs from different origins. Interestingly, the AspDHs from Klebsiella pneumoniae 34618 (KpnAspDH) and Delftia sp. Cs1–4 (DelAspDH) showed successful soluble expression. KpnAspDH and DelAspDH containing C-terminal hexa-histidine tags were purified and characterized for their catalytic properties. Notably, in addition to its high reductive amination activity, DelAspDH exhibited considerable stability as compared to the other source of AspDHs. This work thus provides novel enzyme resource for engineering strains capable of producing AFAAs under anaerobic conditions.

To4, the first Tityus obscurus β-toxin fully electrophysiologically characterized on human sodium channel isoforms

Many scorpion toxins that act on sodium channels (NaScTxs) have been characterized till date. These toxins may act modulating the inactivation or the activation of sodium channels and are named α- or β-types, respectively. Some venom toxins from Tityus obscurus (Buthidae), a scorpion widely distributed in the Brazilian Amazon, have been partially characterized in previous studies; however, little information about their electrophysiological role on sodium ion channels has been published. In the present study, we describe the purification, identification and electrophysiological characterization of a NaScTx, which was first described as Tc54 and further fully sequenced and renamed To4. This toxin shows a marked β-type effect on different sodium channel subtypes (hNav1.1–hNav1.7) at low concentrations, and has more pronounced activity on hNav1.1, hNav1.2 and hNav1.4. By comparing To4 primary structure with other Tityus β-toxins which have already been electrophysiologically tested, it is possible to establish some key amino acid residues for the sodium channel activity. Thus, To4 is the first toxin from T. obscurus fully electrophysiologically characterized on different human sodium channel isoforms.

Assessment of dual inhibitory activity of epifriedelanol isolated from <i>Cayratia trifolia</i> against ovarian cancer

<p class="Abstract"><em>Cayratia trifolia</em> is used as diuretic, in tumors, neuralgia and splenopathy. However, compounds depicting anti-ovarian cancer activities from this plant source have not yet been identified and structurally characterized till date. In the present study, X-ray structure of epifriedelanol, a bioactive compound, isolated from the ethanolic extract of the <em>C. trifolia</em> and its binding affinities against a few proteins (HER2, EGFR and CXCR4) that are reported to get overexpressed under ovarian cancer had been thoroughly studied by using molecular docking means. Binding affinities of the compound vis-à-vis that of carboplatin, a FDA approved drug to the ovarian cancer, to interact with the protein targets are quite impressive. The drug-likeness properties of the epifriedelanol and scope to develop the compound as a potent anti-ovarian cancer drug are discussed in this paper.</p><p> </p>

Diversity of Globins in Myxobacteria

Globins are heme proteins that are capable of reversible oxygen binding. All globins can be classified into three families: the M (myoglobin - like), S (sensor) and T (truncated) globins. M and S globins exhibit the canonical 3/3 α - helical fold, and T globins are characterized by a 2/2 α - he lical fold. Globins in the genomes of myxobacteria have not been characterized till date. Myxobacteria have very large genomes relative to other bacteria and have a unique life cycle that involves the aggregation of cells into fruiting bodies under starvat ion conditions. The diversity of globin like sequences in 14 sequenced genomes of myxobacteria is presented in this review. In myxobacterial globins some unusual domain architectures are identified that have not been characterized in bacteria so far; these are: i) a unique chimeric group I 2/2 HbN in the genome of Corallococcus coralloides DSM 2259; ii) M globin chimera harboring a central and a C - terminal globin domain in Sorangium cellulosum ‘so ce 56’ and Plesiocystis pacifica SIR - 1 respectively; iii) tw o tandem globin domains on the same M globin polypeptide in the genomes of Sorangium cellulosum .

Molecular tools for deciphering the microbial community structure and diversity in rumen ecosystem

Rumen microbial community comprising of bacteria, archaea, fungi, and protozoa is characterized not only by the high population density but also by the remarkable diversity and the most complex microecological interactions existing in the biological world. This unprecedented biodiversity is quite far from full elucidation as only about 15-20% of the rumen microbes are identified and characterized till date using conventional culturing and microscopy. However, the last two decades have witnessed a paradigm shift from cumbersome and time-consuming classical methods to nucleic acid-based molecular approaches for deciphering the rumen microbial community. These techniques are rapid, reproducible and allow both the qualitative and quantitative assessment of microbial diversity. This review describes the different molecular methods and their applications in elucidating the rumen microbial community.