Deciphering the mannose transfer mechanism of mycobacterial PimE by molecular dynamics simulations.

Phosphatidyl-myo-inositol mannosides (PIMs), Lipomannan (LM), and Lipoarabinomannan (LAM) are essential components of the cell envelopes of mycobacteria. At the beginning of the biosynthesis of these compounds, phosphatidylinositol (PI) is mannosylated and acylated by various enzymes to produce Ac1/2PIM4, which is used to synthesize either Ac1/2PIM6 or LM/LAM. The protein PimE, a membrane-bound glycosyltransferase (GT-C), catalyzes the addition of a mannose group to Ac1PIM4 to produce Ac1PIM5, using polyprenolphosphate mannose (PPM) as the mannose donor. PimE-deleted Mycobacterium smegmatis (Msmeg) showed structural deformity and increased antibiotic and copper sensitivity. Despite knowing that the mutation D58A caused inactivity in Msmeg, how PimE catalyzes the transfer of mannose from PPM to Ac1/2PIM4 remains unknown. In this study, analyzing the AlphaFold structure of PimE revealed the presence of a tunnel through the D58 residue with two differently charged gates. Molecular docking suggested PPM binds to the hydrophobic tunnel gate, whereas Ac1PIM4 binds to the positively charged tunnel gate. Molecular dynamics (MD) simulations further demonstrated the critical roles of the residues N55, F87, L89, Y163, Q165, K197, L198, R251, F277, W324, H326, and I375 in binding PPM and Ac1PIM4. The mutation D58A caused a faster release of PPM from the catalytic tunnel, explaining the loss of PimE activity. Along with a hypothetical mechanism of mannose transfer by PimE, we also observe the presence of tunnels through a negatively charged aspartate or glutamate with two differently-charged gates among most GT-C enzymes. Common hydrophobic gates of GT-C enzymes probably harbor sugar donors, whereas, differently-charged tunnel gates accommodate various sugar-acceptors.

Crystal structure of a thiolase from Archaeal Pyrococcus furiosus and its in silico functional annotation.

In most of the eukaryotes and archaea, isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DMAPP) essential building blocks of all isoprenoids synthesized in the mevalonate pathway. Here, the first enzyme of this pathway, acetoacetyl CoA thiolase (PFC_04095) from an archaea Pyrococcus furiosus is structurally characterized. The crystal structure of PFC_04095 is determined at 2.7 Å resolution, and the crystal structure reveals the absence of catalytic acid/base cysteine in its active site, which is uncommon in thiolases. In place of cysteine, His285 of HDAF motif performs both protonation and abstraction of proton during the reaction. The crystal structure shows that the distance between Cys83 and His335 is 5.4 Å. So, His335 could not abstract a proton from nucleophilic cysteine (Cys83), resulting in the loss of enzymatic activity of PFC_04095. MD simulations of the docked PFC_04095-acetyl CoA complex show substrate binding instability to the active site pocket. Here, we have reported that the stable binding of acetyl CoA to the PFC_04095 pocket requires the involvement of three protein complexes, i.e., thiolase (PFC_04095), DUF35 (PFC_04100), and HMGCS (PFC_04090).

Tuneable quantised conductance memory states in TiO2based resistive switching devices in crossbar geometry for high density memory applications.

The tunability and controllability of conductance quantization mediated multilevel resistive switching (RS) memory devices, fabricated in crossbar geometry can be a promising alternative for boosting storage density. Here, we report fabrication of Cu/TiO2/Pt based RS devices in 8 × 8 crossbar geometry, which showed reliable bipolar RS operations. The crossbar devices showed excellent spatial and temporal variability, time retention and low switching voltage (<1 V) and current (∼100µA). Furthermore, during the reset switching, highly repeatable and reliable integral and half-integral quantized conductance (QC) was observed. The observed QC phenomenon was attributed to the two dimensional confinement of electrons as lateral width of the conducting filament (CF) matches the fermi wavelength. The magnitude and number of the QC steps were found to increase from ∼2.5 to 12.5 and from 5 to 18, respectively by increasing the compliance current (IC) from 50 to 800µA which also increased the diameter of the CF from ∼1.2 to 3.3 nm. The enhancement in both number and magnitude of QC states was explained using electrochemical dissolution mechanism of CF of varying diameter. A thicker CF, formed at higherIC, undergoes a gradual rupture during reset process yielding a greater number of QC steps compared to a thinner CF. The realisation of QC states in the crossbar Cu/TiO2/Pt device as well asICmediated tunability of their magnitude and number may find applications in high-density resistive memory storage devices and neuromorphic computing.

Edema India: Executive Summary of Screening, Diagnosis, and Management of Edema.

The management of edema requires a systematic approach to screening, diagnosis, and treatment, with an essential initial assessment to differentiate between generalized and localized edema. The Association of Physicians of India (API) aimed to develop the first Indian Edema Consensus (Edema India), offering tailored recommendations for screening, diagnosing, and managing edema based on the insights from the expert panel. The panel suggested when evaluating edema symptoms, important factors to consider include the patient's current illness, medical history, risk factors, family history, and medications. Key diagnostic investigations for edema include complete blood count, cardiovascular imaging and markers, deep vein thrombosis (DVT) assessment, along with renal, hepatic, and thyroid function tests. Edema management involves a combination of pharmacologic and nonpharmacologic interventions, including limb elevation, physiotherapy, compression therapy, fluid removal, diuretics (loop diuretics: first-line therapy), and a sodium-restricted diet. The panel believed that educating patients could foster a preventive mindset, helping to prevent the worsening of edema.

Crystal structure of a mycobacterial secretory protein Rv0398c and in silico prediction of its export pathway.

Secretory proteins are used by pathogenic bacteria to manipulate the host systems and compete with other microorganisms, thereby enabling their survival in their host. Similar to other bacteria, secretory proteins of Mycobacterium tuberculosis also play a pivotal role in evading immune response within hosts, thereby leading to acute and latent tuberculosis infection. Prokaryotes have several classes of bacterial secretory systems out of which the Sec and Tat pathways are the most conserved in Mtb to transport proteins across the cytoplasmic membrane. Here, we report the crystal structure of a secretory protein, Rv0398c determined to 1.9 Å resolution. The protein comprises a core of antiparallel ß sheets surrounded by α helices adopting a unique ß sandwich fold. Structural comparison with other secretory proteins in Mtb and other pathogenic bacteria reveals that Rv0398c may be secreted via the Sec pathway. Our structural and in silico analyses thus provide mechanistic insights into the pathway adopted by Mtb to transport out secretory protein, Rv0398c which will facilitate the invasion to the host immune system.

Staphylococcal superantigen-like protein 10 enhances the amyloidogenic biofilm formation in Staphylococcus aureus.

Staphylococcus aureus is a highly infectious pathogen that represents a significant burden on the current healthcare system. Bacterial attachment to medical implants and host tissue, and the establishment of a mature biofilm, play an important role in chronic diseases such as endocarditis, osteomyelitis and wound infections. These biofilms decrease bacterial susceptibility to antibiotics and immune defences, making the infections challenging to treatment. S. aureus produces numerous exotoxins that contribute to the pathogenesis of the bacteria. In this study, we have identified a novel function of staphylococcal superantigen-like protein 10 (SSL10) in enhancing the formation of staphylococcal biofilms. Biofilm biomass is significantly increased when SSL10 is added exogenously to bacterial cultures, whereas SSL2 and SSL12 are found to be less active. Exogenously added SSL10 mask the surface charge of the bacterial cells and lowers their zeta potential, leading to the aggregation of the cells. Moreover, the biofilm formation by SSL10 is governed by amyloid aggregation, as evident from spectroscopic and microscopic studies. These findings thereby give the first overview of the SSL-mediated amyloid-based biofilm formation and further drive the future research in identifying potential molecules for developing new antibacterial therapies against Staphylococcus aureus.

Tunable, reversible resistive switching behavior of PVA-zirconia nanocomposite films and validation of the trap-assisted switching mechanism by the selective application of external bias voltages.

Flexible, free-standing polyvinyl alcohol (PVA)-zirconia (mean particle size ∼24 nm) nanocomposite films have been synthesized and their performance as a potential next-generation resistive switching device material has been assessed in this report. The nanocomposite films switch from a high resistive state (HRS) to a low resistive state (LRS) at the SET potential and from LRS to HRS at the RESET potential within the voltage window of 5 V. The origination of trap-assisted SET/RESET potentials has been experimentally validated by analyzing the experimental data and invoking various theoretical models. The impregnation of zirconium dioxide (ZrO2) nanoparticles considerably enhances the interfacial charges facilitated by the formation of dangling bonds. The current (I)-voltage (V) characteristics elucidate how the alteration of free volumetric space in the nanocomposites can modify the SET-RESET potential. This leads to tunable SET/RESET potential, good resistance ratio (∼80), and extensive cycling ability of these PVA-ZrO2 organic flexible nanocomposite films. Herein, we have also investigated the effect of applying external bias voltage (equal to the RESET potential) for possible energy bandgap modification and polymer chain orientation. The impedance spectra differ considerably when the sample is subjected to SET, RESET, and zero voltage bias. The observations have been correlated with the UV-vis absorption spectra and electrical studies. The adopted analysis method and obtained results can open up new avenues for designing and analyzing resistive switching-based random-access memory devices.

Rationally Designed Novel Phenyloxazoline Synthase Inhibitors: Chemical Synthesis and Biological Evaluation to Accelerate the Discovery of New Antimycobacterial Antibiotics.

The uncontrolled spread of drug-resistant tuberculosis (DR-TB) clinical cases necessitates the urgent discovery of newer chemotypes with novel mechanisms of action. Here, we report the chemical synthesis of rationally designed novel transition-state analogues (TSAs) by targeting the cyclization (Cy) domain of phenyloxazoline synthase (MbtB), a key enzyme of the conditionally essential siderophore biosynthesis pathway. Following bio-assay-guided evaluation of TSA analogues preferentially in iron-deprived and iron-rich media to understand target preferentiality against a panel of pathogenic and non-pathogenic mycobacteria strains, we identified a hit, i.e., TSA-5. Molecular docking, dynamics, and MMPBSA calculations enabled us to comprehend TSA-5's stable binding at the active site pocket of MbtB_Cy and the results imply that the MbtB_Cy binding pocket has a strong affinity for electron-withdrawing functional groups and contributes to stable polar interactions between enzyme and ligand. Furthermore, enhanced intracellular killing efficacy (8 µg/mL) of TSA-5 against Mycobacterium aurum in infected macrophages is noted in comparison to moderate in vitro antimycobacterial efficacy (64 µg/mL) against M. aurum. TSA-5 also demonstrates whole-cell efflux pump inhibitory activity against Mycobacterium smegmatis. Identification of TSA-5 by focusing on the modular MbtB_Cy domain paves the way for accelerating novel anti-TB antibiotic discoveries.

Role of regional diffusion tensor imaging (DTI)-derived tensor metrics in the evaluation of intracranial gliomas and its histopathological correlation.

Background: The imaging of brain tumours has significantly improved with the use of advanced magnetic resonance (MR) techniques like diffusion tensor imaging (DTI). This study was conducted to analyse the utility of DTI-derived tensor metrics in the evaluation of intracranial gliomas with histopathological correlation and further adoption of these image-data analyses in clinical setting. Methods: A total of 50 patients with suspected diagnosis of intracranial gliomas underwent DTI along with conventional MR examination. The study correlated various DTI parameters in the enhancing part of the tumour and the peritumoral region with the histopathological grades of the intracranial gliomas. Results: The study revealed higher values of Cl (linear anisotropy), Cp (planar anisotropy), AD (axial diffusivity), FA (fractional anisotropy) and RA (relative anisotropy) and lower values of Cs (spherical anisotropy), MD (mean diffusivity) and RD (radial diffusivity) in the enhancing part of the tumour in case of high-grade gliomas. However, in the peritumoral region, the values of Cl, Cp, AD, FA and RA were less whereas values of Cs, MD and RD were more in high-grade gliomas than in the low-grade gliomas. The various cutoff values of these DTI-derived tensor metrics were found to be statistically significant. Conclusion: DTI-derived tensor metrics can be a valuable tool in differentiation between high-grade and low-grade gliomas which might be accepted in clinical practice in near future.

In-silico identification of critical residues in the mannose-transfer mechanism of phosphatidyl-myo-inositol mannosyltransferase B'.

The complex cellular envelope is one of the major reasons behind the survival in hostile conditions and the emergence of the drug-resisting properties of mycobacteria. Phosphatidyl-myo-inositol hexamannoside (PIM6), Lipomannan (LM), and Lipoarabinomannan (LAM) are important structural constituents of the cell envelope and have roles in modulating host immune functions. Phosphatidyl-myo-inositol (PI) is first mannosylated at the 2-position of the inositol group by phosphatidyl-myo-inositol mannosyltransferase A (PimA) to produce phosphatidyl-myo-inositol monomannoside (PIM1). This PIM1 is then further mannosylated at the 6-position of the inositol group by phosphatidyl-myo-inositol mannosyltransferase B' (PimB') utilizing GDP-mannose as the mannose-donor to synthesize phosphatidyl-myo-inositol dimannoside (PIM2) and GDP. Further mannosylation and acylation on PIM2 produce Ac1/2PIM4, which can then be converted to either Ac1/2PIM6 or LM/LAM. Detailed functional mechanism of how PimB' transfers the mannose sugar to PIM1 is not understood. Using molecular docking, the interactions of PimB' with the substrate PIM1 and the product PIM2 are analyzed here. Molecular dynamics (MD) simulations of PimB' with the substrates and the products were performed for 300ns to find out critical residues involved in the mannose-transfer reaction. Docking and MD analyses indicated the residues R206 and R210 bind both PIM1 and PIM2 and are critical in the mannose-transfer reaction. The residues 120HEVGWSMLPGS130 and 281RTRGGGL288 were involved in the transfer of PIM1 from the active site. The residues 18IGG20, K211, E290, G291, 294IV295, and E298 were also important in the mannosylation reaction. The crucial residues obtained from this study may help design novel drugs against mycobacterial PimB'.

A subtractive proteomics and immunoinformatics approach towards designing a potential multi-epitope vaccine against pathogenic Listeriamonocytogenes.

Listeria monocytogenes is the causative agent of listeriosis, which is dangerous for pregnant women, the elderly or individuals with a weakened immune system. Individuals with leukaemia, cancer, HIV/AIDS, kidney transplant and steroid therapy suffer from immunological damage are menaced. World Health Organization (WHO) reports that human listeriosis has a high mortality rate of 20-30% every year. To date, no vaccine is available to treat listeriosis. Thereby, it is high time to design novel vaccines against L. monocytogenes. Here, we present computational approaches to design an antigenic, stable and safe vaccine against the L. monocytogenes that could help to control the infections associated with the pathogen. Three vital pathogenic proteins of L. monocytogenes, such as Listeriolysin O (LLO), Phosphatidylinositol-specific phospholipase C (PI-PLC), and Actin polymerization protein (ActA), were selected using a subtractive proteomics approach to design the multi-epitope vaccine (MEV). A total of 5 Cytotoxic T-lymphocyte (CTL) and 9 Helper T-lymphocyte (HTL) epitopes were predicted from these selected proteins. To design the multi-epitope vaccine (MEV) from the selected proteins, CTL epitopes were joined with the AAY linker, and HTL epitopes were joined with the GPGPG linker. Additionally, a human ß-defensin-3 (hBD-3) adjuvant was added to the N-terminal side of the final MEV construct to increase the immune response to the vaccine. The final MEV was predicted to be antigenic, non-allergen and non-toxic in nature. Physicochemical property analysis suggested that the MEV construct is stable and could be easily purified through the E. coli expression system. This in-silico study showed that MEV has a robust binding interaction with Toll-like receptor 2 (TLR2), a key player in the innate immune system. Current subtractive proteomics and immunoinformatics study provides a background for designing a suitable, safe and effective vaccine against pathogenic L. monocytogenes.

Stochastic gene transcription with non-competitive transcription regulatory architecture.

The transcription factors, such as activators and repressors, can interact with the promoter of gene either in a competitive or non-competitive way. In this paper, we construct a stochastic model with non-competitive transcriptional regulatory architecture and develop an analytical theory that re-establishes the experimental results with an improved data fitting. The analytical expressions in the theory allow us to study the nature of the system corresponding to any of its parameters and hence, enable us to find out the factors that govern the regulation of gene expression for that architecture. We notice that, along with transcriptional reinitiation and repressors, there are other parameters that can control the noisiness of this network. We also observe that, the Fano factor (at mRNA level) varies from sub-Poissonian regime to super-Poissonian regime. In addition to the aforementioned properties, we observe some anomalous characteristics of the Fano factor (at mRNA level) and that of the variance of protein at lower activator concentrations in the presence of repressor molecules. This model is useful to understand the architecture of interactions which may buffer the stochasticity inherent to gene transcription.

A Study on Clinical Profile of Hemifacial Spasm in India and the Therapeutic Response to Botulinum Toxin Type A Injection as well as Pre and Postinjection Quality of Life.

BACKGROUND: Hemifacial spasm (HFS) is a distressing, involuntary, irregular tonic-clonic contraction of the facial muscles innervated by the seventh cranial nerve. It affects the quality of life. Botulinum toxin is a preferred symptomatic treatment option for the condition. However, there is a lack of study in the Indian scenario. Therefore, we observed the demographic profile, clinical spectrum, therapeutic response, and adverse effects of botulinum toxin and assessed the quality of life in the pre and postinjection phases in our subjects with HFS. MATERIALS AND METHODS: The study design is a prospective open-label observational study. Consecutive cases of HFS were selected from the general neurology outpatient department (OPD) and movement disorder clinic of a medical college hospital in Eastern India. Clinical and relevant neuroimaging studies excluded mimickers and secondary causes of HFS. Institutional Ethics Committee's permission was obtained. Informed consent was taken from patients before botulinum toxin injection. The pre and postinjection assessment tools were spasm rate for a specific period of time, quantification of facial asymmetry, widening palpebral fissure by visual analog scale, Jankovic disability rating scale, HFS-7 scale, and videography. RESULTS: A total of 250 cases of HFS (F:M = 138:112) were studied. The mean age of presentation was 47 years. The mean dose of botulinum toxin injection was 24.2 units per patient. The mean duration of improvement was 4 months. The spasm frequency was decreased by 90%, and the facial asymmetry was improved by 86%. The improvement in quality of life was 86%. Local adverse effects are seen in 10.4% of cases, and all were reversible. CONCLUSION: This is one of the largest studies on the effects of botulinum toxin in subjects with HFS in the Indian population. Periodic injection of botulinum toxin is a safe and effective therapy for subjects with HFS. There is a significant improvement in the quality of life following botulinum toxin therapy in subjects with HFS. Adverse effects were local, mild, well-tolerated, and reversible.

Dual Functionalized Injectable Hybrid Extracellular Matrix Hydrogel for Burn Wounds.

Low strength and rapid biodegradability of acellular dermal matrix (ADM) restrict its wider clinical application as a rapid cell delivery platform in situ for management of burn wounds. Herein, the extracted ADM was modified by a dual cross-linking approach with ionic crosslinking using chitosan and covalent cross-linking using an iodine-modified 2,5-dihydro-2,5-dimethoxy-furan cross-linker, termed as CsADM-Cl. In addition, inherent growth factors and cytokines were found to be preserved in CsADM-Cl, irrespective of ionic/covalent crosslinking. CsADM-Cl demonstrated improvement in post crosslinking stiffness with a decreased biodegradation rate. This hybrid crosslinked hydrogel supported adhesion, proliferation, and migration of human foreskin-derived fibroblasts and keratinocytes. Also, the angiogenic potential of CsADM-Cl was manifested by chick chorioallantoic membrane assay. CsADM-Cl showed excellent antibacterial activity against Escherichia coli and Staphylococcus aureus. Moreover, CsADM-Cl treated full thickness burn wounds and demonstrated rapid healing marked with superior angiogenesis, well-defined dermal-epidermal junctions, mature basket weave collagen deposition, and development of more pronounced secondary appendages. Altogether, the bioactive CsADM-Cl hydrogel established significant clinical potential to support wound healing as an apt injectable antibacterial matrix to encounter unmet challenges concerning critical burn wounds.

Structure-based Epitope Mapping of Mycobacterium tuberculosis Secretary Antigen MTC28.

Secretary proteins of Mycobacterium tuberculosis are key players of the mycobacterial infection pathway. MTC28 is a 28-kDa proline-rich secretary antigen of Mycobacterium tuberculosis and is only conserved in pathogenic strains of mycobacteria. Here we report the crystal structure of MTC28 at 2.8- and 2.15-Å resolutions for the structure-based epitope design. MTC28 shares a "mog1p"-fold consisting of seven antiparallel ß strands stacked between α helices. Five probable epitopes have been located on a solvent-accessible flexible region by computational analysis of the structure of MTC28. Simultaneously, the protein is digested with trypsin and the resulting fragments are purified by HPLC. Such 10 purified peptide fragments are screened against sera from patients infected with pulmonary tuberculosis (PTB). Two of these 10 fragments, namely (127)ALDITLPMPPR(137) and (138)WTQVPDPNVPDAFVVIADR(156),are found to be major immunogenic epitopes that are localized on the outer surface of the protein molecule and are part of a single continuous epitope that have been predicted in silico Mutagenesis and antibody inhibition studies are in accordance with the results obtained from epitope mapping.

Enediyne-based protein capture agents: demonstration of an enediyne moiety acting as a photoaffinity label.

Two enediyne based protein-capture compounds 1 and 2 were synthesized. Both these molecules have an aryl sulfonamide for reversible binding with Human Carbonic Anhydrase II (HCA II) and a pyrene moiety for the visualization of a capture event. While compound 1 has an aryl azide as a photo cross-linking agent, compound 2 lacks the azide moiety. Capture experiments with HCA II however show that both 1 and 2 can photo cross-link with the protein as indicated in gel electrophoresis as well as MALDI analysis after tryptic digestion of HCA II. This observation demonstrates the ability of the enediyne moiety to act as a photo-affinity label possibly via the addition of nucleophilic amino acids to the partially zwitterionic singlet form of the diradical generated by photo Bergman cyclization.

Molecular insights into RNA-binding properties of Escherichia coli-expressed RNA-dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus.

Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) is responsible for morbidity of the Indian non-mulberry silkworm, A. mylitta. AmCPV belongs to the family Reoviridae and has 11 double-stranded (ds) RNA genome segments (S1-S11). Segment 2 (S2) encodes a 123-kDa polypeptide with RNA-dependent RNA polymerase (RdRp) activity. To examine the RNA-binding properties of the viral polymerase, the full-length RdRp and its three domains (N-terminal, polymerase and C-terminal domains) were expressed in Escherichia coli BL21 (DE3) cells with hexahistidine and trigger factor tag fused consecutively at its amino terminus, and the soluble fusion proteins were purified. The purified full-length polymerase specifically bound to the 3' untranslated region (3'-UTR) of a viral plus-sense (+) strand RNA with strong affinity regardless of the salt concentrations, but the isolated polymerase domain of the enzyme exhibited poor RNA-binding ability. Further, the RdRp recognition signals were found to be different from the cis-acting signals that promote minus-sense (-) strand RNA synthesis, because different internal regions of the 3'-UTR of the (+) strand RNA did not effectively compete out the binding of RdRp to the intact 3'-UTR of the (+) strand RNA, but all of these RNA molecules could serve as templates for (-) strand RNA synthesis by the polymerase.

Green approach to photoluminescent carbon dots for imaging of gram-negative bacteria Escherichia coli.

Fluorescent carbon dots, zero-dimensional nanomaterials with surface ligands, have been studied extensively over the past few years in biolabelling or fluorescence-based live cell assays. In the past, synthetic organic dyes have been used as cell tracking materials, but they have severe limitations; fluorescent carbon dots may pave the way to biolabelling and cell imaging. In this work, green fluorescent carbon dots have been synthesized from a green source, gram, without any sort of covalent or ionic modifications. These gram-derived carbon dots are unique with respect to synthetic commercial cell-tracking dyes as they are non-toxic, cell internalization occurs quickly, and they have excellent bioconjugation with bacterial cells. Our aim is to establish these carbon dots in a biolabelling assay with its other physicochemical features like the tunable luminescence property, high degree of water solubility and low toxicity, towards various environments (wide range of pH, high ionic strength). Our study introduces a new perspective on the commercialization of carbon dots as a potential alternative to synthetic organic dyes for fluorescence-based cell-labelling assays.

Reconstitution of the RNA-dependent RNA polymerase activity of Antheraea mylitta cypovirus in vitro using separately expressed different functional domains of the enzyme.

Antheraea mylitta cytoplasmic polyhedrosis virus is a segmented dsRNA virus of the family Reoviridae. Segment 2 (S2)-encoded RNA-dependent RNA polymerase (RdRp) helps the virus to propagate its genome in the host cell of the silkworm, Antheraea mylitta. Cloning, expression, purification and functional analysis of individual domains of RdRp have demonstrated that the purified domains interact in vitro. The central polymerase domain (PD) shows nucleotide binding properties, but neither the N-terminal domain (NTD) nor the C-terminal domain (CTD). Isolated PD does not exhibit RdRp activity but this activity can be reconstituted when all three domains are included in the reaction mixture. Molecular dynamics simulation suggests that the isolated PD has increased internal motions in comparison to when it is associated with the NTD and CTD. The motions of the separated PD may lead to the formation of a less accessible RNA template-binding channel and, thus, impair RdRp activity.

Crystal structure of dehydratase component HadAB complex of mycobacterial FAS-II pathway.

Fatty acid biosynthesis type II in mycobacteria delivers the fatty acids required for mycolic acid synthesis. The pathway employs a unique maoC like ß-hydroxyacyl-ACP dehydratase HadAB or HadBC heterodimer in the third step of the elongation cycle. Here we report the crystal structure of the HadAB complex determined using a Pb-SIRAS method. Crystal structure aided with enzymatic study establishes the roles of HadA as a scaffolding component and HadB as a catalytic component together indispensable for the activity. The detailed structural analysis of HadAB in combination with MD simulation endorses the spatial orientation of the central hot-dog helix and the dynamic nature of its associated loop in regulation of substrate specificities in dehydratase/hydratase family enzymes.