02223nas a2200205 4500008004100000245011400041210006900155260000800224300001200232490000700244520150600251100001701757700001801774700002101792700002201813700001601835700001701851700002501868856012401893 2016 eng d00aMolecular cloning and in silico studies of physiologically significant trehalase from drosophila melanogaster0 aMolecular cloning and in silico studies of physiologically signi cNOV a282-2920 v923 aTrehalase, a physiologically important glycosidase is known for its crucial role in insect glycometabolism and stress recovery. The present study describes the molecular cloning of a gene fragment, encoding the catalytically active trehalase from Drosophila melanogaster (DmTre) and its heterologous expression in Escherichia coli. The 1275 bp gene was overexpressed in two different vectors viz., pET28a and pCOLD TF and investigated for variable soluble expression, purification and activity of the recombinant enzyme with optimum pH and temperature of enzyme as 6 and 55 degrees C, respectively. The sequence was characterized in silico by subjecting it to homology search, multiple sequence alignment and phylogenetic tree construction revealing its identity to other trehalases which belong to glycoside hydrolase family 37. The deduced amino acid sequence and modeled 3D structure of DmTre possessed all features of trehalase superfamily, including signature motifs and catalytic domain. The active site pocket of recombinant DmTre was compared with the crystal structure of E. coli trehalase identifying Glu424 and Asp226 as the putative catalytic residues. Additionally, enzyme-substrate docking suggests possible involvement of other residues in the catalysis along with Asp226. The present study holds significance in understanding the structural aspects of Drosophila trehalase in spite of unavailabilty of eukaryotic trehalase crystal structure. (C) 2016 Elsevier B.V. All rights reserved.1 aShukla, Ekta1 aThorat, Leena1 aBhavnani, Varsha1 aBendre, Ameya, D.1 aPal, J., K.1 aNath, B., B.1 aGaikwad, Sushama, M. uhttp://library.ncl.res.in/content/molecular-cloning-and-silico-studies-physiologically-significant-trehalase-drosophila01871nas a2200157 4500008004100000245009900041210006900140260000800209300001200217490000800229520128800237100002201525700002601547700001901573856012101592 2018 eng d00aAnalysis of Kunitz inhibitors from plants for comprehensive structural and functional insights0 aAnalysis of Kunitz inhibitors from plants for comprehensive stru cJUL a933-9430 v1133 aLegume Kunitz type trypsin inhibitor (KTI) family is one of the most versatile families of proteins. A typical KTI features a single peptide folded in beta-trefoil manner, with the molecular weight about 20-22 kDa and two disulphide bonds. The members are known to inhibit a wide range of serpins proteases at the same time many of them possess unique features. Copaifera langsdoiffii Trypsin inhibitor (CTI) has a beta-trefoil fold made up of two non-covalently bound polypeptide chains with only a single disulfide bridge. Delonix regia Trypsin inhibitor (DrTI) has one amino acid insertion between P1 and P2 of the reactive site distorting its conformation. Bauhinia bauhinioides Cruzipain inhibitor (BbCI) has a conservative beta-trefoil fold but lacks disulfide bonds. Such subtle differences in structures make Kunitz inhibitors different from other inhibitor families. Most of the studies on these inhibitors are focused towards their proposed role in defense from insect pests and wounding but their exact physiological role in nature is still uncharted. Thus, it would be very interesting to closely analyze the structural details of these inhibitors in order to ascertain their biological role and other fascinating applications. (C) 2018 Elsevier B.V. All rights reserved.1 aBendre, Ameya, D.1 aRamasamy, Sureshkumar1 aSuresh, C., G. uhttp://library.ncl.res.in/content/analysis-kunitz-inhibitors-plants-comprehensive-structural-and-functional-insights02643nas a2200277 4500008004100000022001400041245011100055210006900166260000800235300000800243490000600251520174300257653002102000653002302021653001102044653002402055653001402079100001702093700001802110700002202128700002002150700002102170700002402191700002502215856012502240 2018 eng d a2190-572X00aCloning and characterization of trehalase: a conserved glycosidase from oriental midge, Chironomus ramosus0 aCloning and characterization of trehalase a conserved glycosidas cAUG a3520 v83 a

Insect trehalase is a multiferous enzyme, crucial for normal physiological functions as well as under stress conditions. In this report, we present a fundamental study of the trehalase gene segment (1587 bp) from Chironomus ramosus (CrTre) encoding for 529 amino acids, using appropriate bioinformatics tools. C. ramosus, a tropical midge is an emerging animal model to investigate the consequences of environmental stresses. We observed that CrTre belongs to GH family 37 in the CAZy database and possess 57-92% identity to dipteran trehalases. In silico characterization provided information regarding the structural, functional and evolutionary aspects of midge trehalase. In the phylogenetic tree, CrTre clustered with the soluble dipteran trehalases. Moreover, domain functional characterization of the deduced protein sequence by InterProScan (IPR001661), ProSite (PS00927 and PS00928) and Pfam (PF01204) indicated presence of highly conserved signature motifs which are important for the identification of trehalase superfamily. Furthermore, the instability index of CrTre was predicted to be < 40 suggesting its in vivo stability while, the high aliphatic index indicated towards its thermal stability (index value 71-81). The modelled 3D tertiary structure of CrTre depicts a (alpha/alpha)(6) barrel toroidal core. The catalytic domain of the enzyme comprised Glu424 and Asp226 as the putative active site residues. Interestingly, the conserved motifs were observed to be formed by the flexible loopy regions in the tertiary structure. This study revealed essential sequence features of the midge trehalase and offers better insights into the structural aspects of this enzyme which can be correlated with its function.

10aConserved motifs10aHomology modelling10aMidges10aSecondary structure10atrehalose1 aShukla, Ekta1 aThorat, Leena1 aBendre, Ameya, D.1 aJadhav, Santosh1 aPal, Jayanta, K.1 aNath, Bimalendu, B.1 aGaikwad, Sushama, M. uhttp://library.ncl.res.in/content/cloning-and-characterization-trehalase-conserved-glycosidase-oriental-midge-chironomus01832nas a2200241 4500008004100000022001400041245012600055210006900181260000800250300001400258490000700272520099600279653001301275653002201288653002001310653002901330653001201359100002201371700001901393700002801412700002601440856012401466 2019 eng d a0739-110200aStructural insights into the unique inhibitory mechanism of Kunitz type trypsin inhibitor from Cicer arietinum L. Vol. 370 aStructural insights into the unique inhibitory mechanism of Kuni cJUL a2669-26770 v373 a

Kunitz-type trypsin inhibitors bind to the active pocket of trypsin causing its inhibition. Plant Kunitz-type inhibitors are thought to be important in defense, especially against insect pests. From sequence analysis of various Kunitz-type inhibitors from plants, we identified CaTI2 from chickpea as a unique variant lacking the functionally important arginine residue corresponding to the soybean trypsin inhibitor (STI) and having a distinct and unique inhibitory loop organization. To further explore the implications of these sequence variations, we obtained the crystal structure of recombinant CaTI2 at 2.8 angstrom resolution. It is evident from the structure that the variations in the inhibitory loop facilitates non-substrate like binding of CaTI2 to trypsin, while the canonical inhibitor STI binds to trypsin in substrate like manner. Our results establish the unique mechanism of trypsin inhibition by CaTI2, which warrant further research into its substrate spectrum.

10aChickpea10acrystal structure10ainhibitory loop10aKunitz trypsin inhibitor10aTrypsin1 aBendre, Ameya, D.1 aSuresh, C., G.1 aShanmugam, Dhanasekaran1 aRamasamy, Sureshkumar uhttp://library.ncl.res.in/content/structural-insights-unique-inhibitory-mechanism-kunitz-type-trypsin-inhibitor-cicer-002328nas a2200229 4500008004100000022001400041245010400055210006900159260000800228300001200236490000700248520151100255653001301766653003601779653003801815653002501853653002901878100002201907700001701929700002601946856012601972 2020 eng d a1572-388700aFunctional stability and structural transitions of a kunitz trypsin inhibitor from chickpea (CaTI2)0 aFunctional stability and structural transitions of a kunitz tryp cAUG a350-3570 v393 a

Enzymes are important tools for various applications. We have studied structural transitions and functional stability of a Kunitz trypsin inhibitor from Chickpea (CaTI2), a potent insect gut-protease inhibitor, under different stress conditions like non-neutral pH, elevated temperature and co-solvent concentrations. CaTI2 was cloned and expressed in an eukaryotic systemP. pastorisand was investigated for conformational transitions using circular dichroism spectroscopy, differential scanning fluorimetry and activity assay. Native CaTI2 has a sheet dominant structure with 40% beta sheets and possess a single tryptophan residue situated in the hydrophobic core of the enzyme. The recombinant inhibitor maintained its maximum activity under alkaline pH with its secondary structure intact between pH 6-10. CaTI2 was observed to be thermally stable up to 55 degrees C with aT(m) of 61.3 degrees C above which the protein unfolds. On treating with chemical denaturant (urea), the CaTI2 lost its inhibitory potential and native conformation beyond 2 M urea concentration. Moreover, the protein unfolded at lower temperatures as the concentration of denaturant increased, suggesting more complex structural changes. Further, the stability of the inhibitor was found to be directly proportional to the solvent polarity. The data, herein offers significant information of inhibitor stability and activity which could be exploited for its further development into an effective pesticide. [GRAPHICS] .

10aChickpea10aCircular dichroism spectroscopy10aDifferential scanning fluorimetry10aFunctional stability10aKunitz trypsin inhibitor1 aBendre, Ameya, D.1 aShukla, Ekta1 aRamasamy, Sureshkumar uhttp://library.ncl.res.in/content/functional-stability-and-structural-transitions-kunitz-trypsin-inhibitor-chickpea-cati2