01922nas a2200265 4500008004100000022001400041245011800055210006900173260009500242300001200337490000800349520092800357653002401285653001201309653002301321653001501344653001901359653002301378100002901401700002601430700001901456700002401475700002601499856013101525 2013 eng d a0006-291X00aStructural modelling of substrate binding and inhibition in penicillin V acylase from pectobacterium atrosepticum0 aStructural modelling of substrate binding and inhibition in peni a525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USAbACADEMIC PRESS INC ELSEVIER SCIENCEcAUG a538-5430 v4373 a

Penicillin V acylases (PVAs) and bile salt hydrolases (BSHs) have considerable sequence and structural similarity; however, they vary significantly in their substrate specificity. We have identified a PVA from a Gram-negative organism, Pectobacterium atrosepticum (PaPVA) that turned out to be a remote homolog of the PVAs and BSHs reported earlier. Even though the active site residues were conserved in PaPVA it showed high specificity towards penV and interestingly the penV acylase activity was inhibited by bile salts. Comparative modelling and docking studies were carried out to understand the structural differences of the binding site that confer this characteristic property. We show that PaPVA exhibits significant differences in structure, which are in contrast to those of known PVAs and such enzymes from Gram-negative bacteria require further investigation. (C) 2013 Elsevier Inc. All rights reserved.

10aBile salt hydrolase10adocking10aHomology modelling10aInhibition10aPectobacterium10aPenicillin acylase1 aAvinash, Vellore, Sunder1 aPanigrahi, Priyabrata1 aSuresh, C., G.1 aPundle, Archana, V.1 aRamasamy, Sureshkumar uhttps://library.ncl.res.in/content/structural-modelling-substrate-binding-and-inhibition-penicillin-v-acylase-pectobacterium-002536nas a2200193 4500008004100000022001400041245015400055210006900209260011800278300001400396490000800410520169500418100002602113700001602139700001702155700002602172700001902198856012502217 2014 eng d a1350-087200aImproved method for specificity annotation shows a distinct evolutionary divergence among the microbial enzymes of the cholylglycine hydrolase family0 aImproved method for specificity annotation shows a distinct evol aMARLBOROUGH HOUSE, BASINGSTOKE RD, SPENCERS WOODS, READING RG7 1AG, BERKS, ENGLANDbSOC GENERAL MICROBIOLOGYcJUN a1162-11740 v1603 a

Bile salt hydrolases (BSHs) are gut microbial enzymes that play a significant role in the bile acid modification pathway. Penicillin V acylases (PVAs) are enzymes produced by environmental microbes, having a possible role in pathogenesis or scavenging of phenolic compounds in their microbial habitats. The correct annotation of such physiologically and industrially important enzymes is thus vital. The current methods relying solely on sequence homology do not always provide accurate annotations for these two members of the cholylglycine hydrolase (CGH) family as BSH/PVA enzymes. Here, we present an improved method [binding site similarity (BSS)-based scoring system] for the correct annotation of the CGH family members as BSH/PVA enzymes, which along with the phylogenetic information incorporates the substrate specificity as well as the binding site information. The BSS scoring system was developed through the analysis of the binding sites and binding modes of the available BSH/PVA structures with substrates glycocholic acid and penicillin V. The 198 sequences in the dataset were then annotated accurately using BSS scores as BSH/PVA enzymes. The dataset presented contained sequences from Gram-positive bacteria, Gram-negative bacteria and archaea. The clustering obtained for the dataset using the method described above showed a clear distinction in annotation of Gram-positive bacteria and Gram-negative bacteria Based on this clustering and a detailed analysis of the sequences of the CGH family in the dataset, we could infer that the CGH genes might have evolved in accordance with the hypothesis stating the evolution of diderms and archaea from the monoderms.

1 aPanigrahi, Priyabrata1 aSule, Manas1 aSharma, Ranu1 aRamasamy, Sureshkumar1 aSuresh, C., G. uhttps://library.ncl.res.in/content/improved-method-specificity-annotation-shows-distinct-evolutionary-divergence-among-002498nas a2200169 4500008004100000022001400041245019300055210006900248260009100317300001100408490000600419520171600425100001702141700002602158700001902184856012502203 2014 eng d a1932-620300aIn-Silico analysis of binding site features and substrate selectivity in plant flavonoid-3-o glycosyltransferases (F3GT) through molecular modeling, docking and dynamics simulation studies0 aInSilico analysis of binding site features and substrate selecti a1160 BATTERY STREET, STE 100, SAN FRANCISCO, CA 94111 USAbPUBLIC LIBRARY SCIENCEcMAR ae926360 v93 a

Flavonoids are a class of plant secondary metabolites that act as storage molecules, chemical messengers, as well as participate in homeostasis and defense processes. They possess pharmaceutical properties important for cancer treatment such as antioxidant and anti-tumor activities. The drug-related properties of flavonoids can be improved by glycosylation. The enzymes glycosyltransferases (GTs) glycosylate acceptor molecules in a regiospecific manner with the help of nucleotide sugar donor molecules. Several plant GTs have been characterized and their amino acid sequences determined. However, three-dimensional structures of only a few are reported. Here, phylogenetic analysis using amino acid sequences have identified a group of GTs with the same regiospecific activity. The structures of these closely related GTs were modeled using homologous GT structures. Their substrate binding sites were elaborated by docking flavonoid acceptor and UDP-sugar donor molecules in the modeled structures. Eight regions near the acceptor binding site in the N- and C-terminal domain of GTs have been identified that bind and specifically glycosylate the 3-OH group of acceptor flavonoids. Similarly, a conserved motif in the C-terminal domain is known to bind a sugar donor substrate. In certain GTs, the substitution of a specific glutamine by histidine in this domain changes the preference of sugar from glucose to galactose as a result of changed pattern of interactions. The molecular modeling, docking, and molecular dynamics simulation studies have revealed the chemical and topological features of the binding site and thus provided insights into the basis of acceptor and donor recognition by GTs.

1 aSharma, Ranu1 aPanigrahi, Priyabrata1 aSuresh, C., G. uhttps://library.ncl.res.in/content/silico-analysis-binding-site-features-and-substrate-selectivity-plant-flavonoid-3-o-002274nas a2200241 4500008004100000022001400041245014400055210006900199260007900268300000800347490000700355520135400362653002901716653001001745653001001755653002101765100002201786700002901808700002601837700001901863700002101882856012901903 2014 eng d a0141-022900aNew role for penicillin acylases: degradation of acyl homoserine lactone quorum sensing signals by Kluyvera citrophila penicillin G acylase0 aNew role for penicillin acylases degradation of acyl homoserine  a360 PARK AVE SOUTH, NEW YORK, NY 10010-1710 USAbELSEVIER SCIENCE INCcMAR a1-70 v563 a

Use of penicillin acylases for the production of semi-synthetic penicillins is well-known. Escherichia coli penicillin G acylase (EcPGA) has been extensively used for this purpose; however, Kluyvera citrophila penicillin G acylase (KcPGA) is assumed to be a better substitute, owing to its increased resilience to extreme pH conditions and ease of immobilization. In the present article we report a new dimension for the amidase activity of KcPGA by demonstrating its ability to cleave bacterial quorum sensing signal molecules, acyl homoserine lactones (AHL) with acyl chain length of 6-8 with or without oxo-substitution at third carbon position. Initial evidence of AHL degrading capability of KcPGA was obtained using CV026 based bioassay method. Kinetic studies performed at pH 8.0 and 50 degrees C revealed 3-oxo-C6 HSL to be the best substrate for the enzyme with V-max and K-m values of 21.37+0.85 mM/h/mg of protein and 0.1+0.01 mM, respectively. C6 HSL was found to be the second best substrate with V-max. and K-m value of 10.06+0.27 mM/h/mg of protein and 0.28+0.02 mM, respectively. Molecular modeling and docking studies performed on the active site of the enzyme support these findings by showing the fitting of AHLs perfectly within the hydrophobic pocket of the enzyme active site. (c) 2013 Elsevier Inc. All rights reserved.

10aAcyl homoserine lactones10aCV02610aKcPGA10aQuorum quenching1 aMukherji, Ruchira1 aVarshney, Nishant, Kumar1 aPanigrahi, Priyabrata1 aSuresh, C., G.1 aPrabhune, Asmita uhttps://library.ncl.res.in/content/new-role-penicillin-acylases-degradation-acyl-homoserine-lactone-quorum-sensing-signals-000712nas a2200205 4500008004100000022001400041245008800055210006900143260000800212300000900220490000700229653002400236653001600260653002600276100001800302700002600320700002600346700001900372856011500391 2014 eng d a2053-273300aStructure of highly active BSH enzyme with subordinated post-translational excision0 aStructure of highly active BSH enzyme with subordinated posttran cMAR aC2720 v7010aBile salt hydrolase10aMutagenesis10aX-ray crystallography1 aChand, Deepak1 aPanigrahi, Priyabrata1 aRamasamy, Sureshkumar1 aSuresh, C., G. uhttps://library.ncl.res.in/content/structure-highly-active-bsh-enzyme-subordinated-post-translational-excision02373nas a2200193 4500008004100000022001400041245006600055210006600121260009100187300001300278490000700291520167900298100002601977700001602003700002102019700002602040700001902066856009402085 2015 eng d a1932-620300aEngineering proteins for thermostability with iRDP web server0 aEngineering proteins for thermostability with iRDP web server a1160 BATTERY STREET, STE 100, SAN FRANCISCO, CA 94111 USAbPUBLIC LIBRARY SCIENCEcOCT ae01394860 v103 a

Engineering protein molecules with desired structure and biological functions has been an elusive goal. Development of industrially viable proteins with improved properties such as stability, catalytic activity and altered specificity by modifying the structure of an existing protein has widely been targeted through rational protein engineering. Although a range of factors contributing to thermal stability have been identified and widely researched, the in silico implementation of these as strategies directed towards enhancement of protein stability has not yet been explored extensively. A wide range of structural analysis tools is currently available for in silico protein engineering. However these tools concentrate on only a limited number of factors or individual protein structures, resulting in cumbersome and time-consuming analysis. The iRDP web server presented here provides a unified platform comprising of iCAPS, iStability and iMutants modules. Each module addresses different facets of effective rational engineering of proteins aiming towards enhanced stability. While iCAPS aids in selection of target protein based on factors contributing to structural stability, iStability uniquely offers in silico implementation of known thermostabilization strategies in proteins for identification and stability prediction of potential stabilizing mutation sites. iMutants aims to assess mutants based on changes in local interaction network and degree of residue conservation at the mutation sites. Each module was validated using an extensively diverse dataset. The server is freely accessible at http://irdp.ncl.res.in and has no login requirements.

1 aPanigrahi, Priyabrata1 aSule, Manas1 aGhanate, Avinash1 aRamasamy, Sureshkumar1 aSuresh, C., G. uhttps://library.ncl.res.in/content/engineering-proteins-thermostability-irdp-web-server-102495nas a2200277 4500008004100000022001400041245012300055210006900178260010600247300001200353490000800365520149500373653001301868653001801881653001001899653001701909653001201926653001601938100002001954700002601974700002602000700002402026700002002050700002102070856012602091 2015 eng d a0031-942200aMetabolic profiling of chickpea-Fusarium interaction identifies differential modulation of disease resistance pathways0 aMetabolic profiling of chickpeaFusarium interaction identifies d aTHE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, ENGLANDbPERGAMON-ELSEVIER SCIENCE LTDcAUG a120-1290 v1163 a

Chickpea is the third most widely grown legume in the world and mainly used as a vegetarian source of human dietary protein. Fusarium wilt, caused by Fusarium oxysporum f. sp. ciceri (Foc), is one of the major threats to global chickpea production. Host resistance is the best way to protect crops from diseases; however, in spite of using various approaches, the mechanism of Foc resistance in chickpea remains largely obscure. In the present study, non-targeted metabolic profiling at several time points of resistant and susceptible chickpea cultivars using high-resolution liquid chromatography-mass spectrometry was applied to better understand the mechanistic basis of wilt resistance or susceptibility. Multivariate analysis of the data (OPLS-DA) revealed discriminating metabolites in chickpea root tissue after Foc inoculation such as flavonoids, isoflavonoids, alkaloids, amino acids and sugars. Foc inoculated resistant plants had more flavonoids and isoflavonoids along with their malonyl conjugates. Many antifungal metabolites that were induced after Foc infection viz, aurantion-obstine beta-glucosides and querecitin were elevated in resistant cultivar. Overall, diverse genetic and biochemical mechanisms were operational in the resistant cultivar for Foc defense as compared to the susceptible plant. The resistant chickpea plants employed the above-mentioned metabolic pathways as potential defense strategy against Foc. (C) 2015 Elsevier Ltd. All rights reserved.

10aChickpea10aFusarium wilt10aLC-MS10aMetabolomics10aOPLS-DA10aPhytoalexin1 aKumar, Yashwant1 aDholakia, Bhushan, B.1 aPanigrahi, Priyabrata1 aKadoo, Narendra, Y.1 aGiri, Ashok, P.1 aGupta, Vidya, S. uhttps://library.ncl.res.in/content/metabolic-profiling-chickpea-fusarium-interaction-identifies-differential-modulation-002736nas a2200277 4500008004100000022001400041245013100055210006900186260008000255300001400335490000700349520172600356653001702082653002802099653002302127653002102150653001402171653002302185653002002208100002602228700001802254700002202272700002602294700001902320856011902339 2015 eng d a1367-543500aSequence and structure-based comparative analysis to assess, identify and improve the thermostability of penicillin G acylases0 aSequence and structurebased comparative analysis to assess ident aTIERGARTENSTRASSE 17, D-69121 HEIDELBERG, GERMANYbSPRINGER HEIDELBERGcNOV a1493-15060 v423 a

Penicillin acylases are enzymes employed by the pharmaceutical industry for the manufacture of semi-synthetic penicillins. There is a continuous demand for thermostable and alkalophilic enzymes in such applications. We have carried out a computational analysis of known penicillin G acylases (PGAs) in terms of their thermostable nature using various protein-stabilizing factors. While the presence of disulfide bridges was considered initially to screen putative thermostable PGAs from the database, various other factors such as high arginine to lysine ratio, less content of thermolabile amino acids, presence of proline in beta-turns, more number of ion-pair and other non-bonded interactions were also considered for comparison. A modified consensus approach designed could further identify stabilizing residue positions by site-specific comparison between mesostable and thermostable PGAs. A most likely thermostable enzyme identified from the analysis was PGA from Paracoccus denitrificans (PdPGA). This was cloned, expressed and tested for its thermostable nature using biochemical and biophysical experiments. The consensus site-specific sequence-based approach predicted PdPGA to be more thermostable than Escherichia coli PGA, but not as thermostable as the PGA from Achromobacter xylosoxidans. Experimental data showed that PdPGA was comparatively less thermostable than Achromobacter xylosoxidans PGA, although thermostability factors favored a much higher stability. Despite being mesostable, PdPGA being active and stable at alkaline pH is an advantage. Finally, several residue positions could be identified in PdPGA, which upon mutation selectively could improve the thermostability of the enzyme.

10aAlkalistable10abeta-lactam antibiotics10aConsensus approach10aDisulfide bridge10aIon pairs10aPenicillin acylase10aThermostability1 aPanigrahi, Priyabrata1 aChand, Deepak1 aMukherji, Ruchira1 aRamasamy, Sureshkumar1 aSuresh, C., G. uhttps://library.ncl.res.in/content/sequence-and-structure-based-comparative-analysis-assess-identify-and-improve-003098nas a2200289 4500008004100000022001400041245018200055210006900237260006700306300001400373490000700387520197200394653002802366653002902394653002702423653002402450653002402474653001802498100001802516700002702534700002602561700002602587700002502613700002702638700001902665856012402684 2015 eng d a0961-836800aStructure mediation in substrate binding and post-translational processing of penicillin acylases: Information from mutant structures of Kluyvera citrophila penicillin G acylase0 aStructure mediation in substrate binding and posttranslational p a111 RIVER ST, HOBOKEN 07030-5774, NJ USAbWILEY-BLACKWELLcOCT a1660-16700 v243 a

Penicillin acylases are industrially important enzymes for the production of 6-APA, which is used extensively in the synthesis of secondary antibiotics. The enzyme translates into an inactive single chain precursor that subsequently gets processed by the removal of a spacer peptide connecting the chains of the mature active heterodimer. We have cloned the penicillin G acylase from Kluyvera citrophila (KcPGA) and prepared two mutants by site-directed mutagenesis. Replacement of N-terminal serine of the beta-subunit with cysteine (Ser beta 1Cys) resulted in a fully processed but inactive enzyme. The second mutant in which this serine is replaced by glycine (Ser beta 1Gly) remained in the unprocessed and inactive form. The crystals of both mutants belonged to space group P1 with four molecules in the asymmetric unit. The three-dimensional structures of these mutants were refined at resolutions 2.8 and 2.5 angstrom, respectively. Comparison of these structures with similar structures of Escherichia coli PGA (EcPGA) revealed various conformational changes that lead to autocatalytic processing and consequent removal of the spacer peptide. The large displacements of residues such as Arg168 and Arg477 toward the N-terminal cleavage site of the spacer peptide or the conformational changes of Arg145 and Phe146 near the active site in these structures suggested probable steps in the processing dynamics. A comparison between the structures of the processed Serb1Cys mutant and that of the processed form of EcPGA showed conformational differences in residues Arg alpha 145, Phe alpha 146, and Phe beta 24 at the substrate binding pocket. Three conformational transitions of Arg alpha 145 and Phe alpha 146 residues were seen when processed and unprocessed forms of KcPGA were compared with the substrate bound structure of EcPGA. Structure mediation in activity difference between KcPGA and EcPGA toward acyl homoserine lactone (AHL) is elucidated.

10aacyl homoserine lactone10aautocatalytic processing10aconformational changes10aKcPGA_Ser beta 1Cys10aKcPGA_Ser beta 1Gly10aNtn hydrolase1 aChand, Deepak1 aVarshney, NishantKumar1 aRamasamy, Sureshkumar1 aPanigrahi, Priyabrata1 aBrannigan, James, A.1 aWilkinson, Anthony, J.1 aSuresh, C., G. uhttps://library.ncl.res.in/content/structure-mediation-substrate-binding-and-post-translational-processing-penicillin-102652nas a2200373 4500008004100000022001400041245014200055210006900197260006700266300001400333490000700347520139300354653001301747653002301760653001701783653000801800653003101808653001501839100002001854700001701874700002601891700002601917700002001943700002301963700002501986700001602011700001302027700003402040700002402074700002002098700001602118700002102134856012302155 2016 eng d a1467-764400aFusarium oxysporum mediates systems metabolic reprogramming of chickpea roots as revealed by a combination of proteomics and metabolomics0 aFusarium oxysporum mediates systems metabolic reprogramming of c a111 RIVER ST, HOBOKEN 07030-5774, NJ USAbWILEY-BLACKWELLcJUL a1589-16030 v143 a

Molecular changes elicited by plants in response to fungal attack and how this affects plant-pathogen interaction, including susceptibility or resistance, remain elusive. We studied the dynamics in root metabolism during compatible and incompatible interactions between chickpea and Fusarium oxysporum f. sp. ciceri (Foc), using quantitative label-free proteomics and NMR-based metabolomics. Results demonstrated differential expression of proteins and metabolites upon Foc inoculations in the resistant plants compared with the susceptible ones. Additionally, expression analysis of candidate genes supported the proteomic and metabolic variations in the chickpea roots upon Foc inoculation. In particular, we found that the resistant plants revealed significant increase in the carbon and nitrogen metabolism; generation of reactive oxygen species (ROS), lignification and phytoalexins. The levels of some of the pathogenesis-related proteins were significantly higher upon Foc inoculation in the resistant plant. Interestingly, results also exhibited the crucial role of altered Yang cycle, which contributed in different methylation reactions and unfolded protein response in the chickpea roots against Foc. Overall, the observed modulations in the metabolic flux as outcome of several orchestrated molecular events are determinant of plant's role in chickpea-Foc interactions.

10aChickpea10afusarium oxysporum10aMetabolomics10aNMR10aplant-pathogen interaction10aproteomics1 aKumar, Yashwant1 aZhang, Limin1 aPanigrahi, Priyabrata1 aDholakia, Bhushan, B.1 aDewangan, Veena1 aChavan, Sachin, G.1 aKunjir, Shrikant, M.1 aWu, Xiangyu1 aLi, Ning1 aRajmohanan, Pattuparambil, R.1 aKadoo, Narendra, Y.1 aGiri, Ashok, P.1 aTang, Huiru1 aGupta, Vidya, S. uhttps://library.ncl.res.in/content/fusarium-oxysporum-mediates-systems-metabolic-reprogramming-chickpea-roots-revealed02452nas a2200265 4500008004100000022001400041245015600055210006900211260009500280300001000375490000800385520139300393653002201786653002801808653002301836653001601859653002801875100002901903700002601932700001801958700002001976700003401996700002602030856013002056 2016 eng d a1047-847700aStructural analysis of a penicillin V acylase from pectobacterium atrosepticum confirms the importance of two Trp residues for activity and specificity0 aStructural analysis of a penicillin V acylase from pectobacteriu a525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USAbACADEMIC PRESS INC ELSEVIER SCIENCEcFEB a85-940 v1933 a

Penicillin V acylases (PVA) catalyze the deacylation of the beta-lactam antibiotic phenoxymethylpenicillin (Pen V). They are members of the Ntn hydrolase family and possess an N-terminal cysteine as the main catalytic nucleophile residue. They form the evolutionarily related cholylglycine hydrolase (CGH) group which includes bile salt hydrolases (BSH) responsible for bile deconjugation. Even though a few PVA and BSH structures have been reported, no structure of a functional PVA from Gram-negative bacteria is available. Here, we report the crystal structure of a highly active PVA from Gram-negative Pectobacterium atrosepticum (PaPVA) at 2.5 angstrom resolution. Structural comparison with PVAs from Gram-positive bacteria revealed that PaPVA had a distinctive tetrameric structure and active site organization. In addition, mutagenesis of key active site residues and biochemical characterization of the resultant variants elucidated the role of these residues in substrate binding and catalysis. The importance of residue Trp23 and Trp87 side chains in binding and correct positioning of Pen V by PVAs was confirmed using mutagenesis and substrate docking with a 15 ns molecular dynamics simulation. These results establish the unique nature of Gram-negative CGHs and necessitate further research about their substrate spectrum. (C) 2015 Elsevier Inc. All rights reserved.

10aAromatic stacking10aCholylglycine hydrolase10amolecular dynamics10aMutagenesis10aOligomeric interactions1 aAvinash, Vellore, Sunder1 aPanigrahi, Priyabrata1 aChand, Deepak1 aPundle, Archana1 aSuresh, Cheravakattu, Gopalan1 aRamasamy, Sureshkumar uhttps://library.ncl.res.in/content/structural-analysis-penicillin-v-acylase-pectobacterium-atrosepticum-confirms-importance-0