00821nas a2200205 4500008004100000022001400041245012500055210006900180260008100249300001200330490000700342100002000349700002500369700002100394700001500415700002300430700002000453700001900473856012300492 2005 eng d a1744-309100aCloning, preparation and preliminary crystallographic studies of penicillin V acylase autoproteolytic processing mutants0 aCloning preparation and preliminary crystallographic studies of  a9600 GARSINGTON RD, OXFORD OX4 2DQ, OXON, ENGLANDbBLACKWELL PUBLISHINGcJAN a124-1270 v611 aChandra, P., M.1 aBrannigan, James, A.1 aPrabhune, Asmita1 aPundle, A.1 aTurkenburg, J., P.1 aDodson, Guy, G.1 aSuresh, C., G. uhttps://library.ncl.res.in/content/cloning-preparation-and-preliminary-crystallographic-studies-penicillin-v-acylase-001482nas a2200217 4500008004100000022001400041245012500055210006900180260007400249300001200323490000700335520065300342100002100995700001901016700002101035700001801056700002501074700002001099700001901119856012601138 2005 eng d a2053-230X00aCloning, purification, crystallization and preliminary structural studies of penicillin V acylase from Bacillus subtilis0 aCloning purification crystallization and preliminary structural  a2 ABBEY SQ, CHESTER, CH1 2HU, ENGLANDbINT UNION CRYSTALLOGRAPHYcJUL a680-6830 v613 a

{Penicillin acylase proteins are amidohydrolase enzymes that cleave penicillins at the amide bond connecting the side chain to their beta-lactam nucleus. An unannotated protein from Bacillus subtilis has been expressed in Escherichia coli, purified and confirmed to possess penicillin V acylase activity. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 4 M sodium formate in 100 mM Tris-HCl buffer pH 8.2. Diffraction data were collected under cryogenic conditions to a spacing of 2.5 A. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 111.0

1 aRathinaswamy, P.1 aPundle, A., V.1 aPrabhune, Asmita1 aSivaRaman, H.1 aBrannigan, James, A.1 aDodson, Guy, G.1 aSuresh, C., G. uhttps://library.ncl.res.in/content/cloning-purification-crystallization-and-preliminary-structural-studies-penicillin-v-000778nas a2200193 4500008004100000022001400041245010300055210006900158260008100227300001200308490000700320100001900327700002000346700002300366700002200389700002600411700001900437856012800456 2005 eng d a1744-309100aCrystallization and preliminary x-ray characterization of a lectin from Cicer arietinum (chickpea)0 aCrystallization and preliminary xray characterization of a lecti a9600 GARSINGTON RD, OXFORD OX4 2DQ, OXON, ENGLANDbBLACKWELL PUBLISHINGcJAN a141-1430 v611 aKatre, Uma, V.1 aGaikwad, S., M.1 aBhagyawant, S., S.1 aDeshpande, U., D.1 aKhan, Mohammad, Islam1 aSuresh, C., G. uhttps://library.ncl.res.in/content/crystallization-and-preliminary-x-ray-characterization-lectin-cicer-arietinum-chickpea-001747nas a2200169 4500008004100000022001400041245018900055210006900244260007100313300001400384490000700398520099300405100001801398700001501416700001901431856012701450 2005 eng d a0022-326300aDiastereoselective and general route to 5-amino-5-deoxysugars: influence of C-3 substitution on the addition of amines to C-5 of vinyl sulfone-modified Hex-5-enofuranosyl carbohydrates0 aDiastereoselective and general route to 5amino5deoxysugars influ a1155 16TH ST, NW, WASHINGTON, DC 20036 USAbAMER CHEMICAL SOCcSEP a8047-80540 v703 a

In the synthesis of vinyl sulfone-modified hex-5-enofuranosides, the E/Z ratios of the products are influenced by the stereoelectronic property of a group present at the C-3 position. This observation has been utilized to influence the diastereoselectivity of addition of amines to C-5 of vinyl sulfone-modified hex-5-enofuranosides, which are efficient Michael acceptors. The stereoelectronic effect of OMe attached to the beta-face of C-3 (gluco derivative) is sufficient to impose diastereoselectivity overwhelmingly in favor of L-ido-aminosugars when the Michael acceptor is reacted with both primary and secondary amines. 3-O-Benzylated gluco derivative is also effective in producing L-ido-aminosugars but only in reactions with primary amines. The selectivity is lost when an allo derivative with OBn at the alpha-face of C-3 is used. Selected products were desulfonated to establish this new approach as a general and versatile strategy for accessing 5-amino-5-deoxysugars.

1 aDas, Indrajit1 aPathak, T.1 aSuresh, C., G. uhttps://library.ncl.res.in/content/diastereoselective-and-general-route-5-amino-5-deoxysugars-influence-c-3-substitution-000751nas a2200181 4500008004100000022001400041245011900055210006900174260008100243300001200324490000700336100002200343700001900365700001400384700002200398700001800420856013100438 2005 eng d a1744-309100aX-ray crystallographic studies on C-phycocyanins from cyanobacteria from different habitats: marine and freshwater0 aXray crystallographic studies on Cphycocyanins from cyanobacteri a9600 GARSINGTON RD, OXFORD OX4 2DQ, OXON, ENGLANDbBLACKWELL PUBLISHINGcSEP a844-8470 v611 aSatyanarayana, L.1 aSuresh, C., G.1 aPatel, A.1 aMishra, Satyendra1 aGhosh, P., K. uhttps://library.ncl.res.in/content/x-ray-crystallographic-studies-c-phycocyanins-cyanobacteria-different-habitats-marine-and-001733nas a2200229 4500008004100000022001400041245015100055210006900206260007900275300001000354490000700364520082100371653002501192653002201217653002601239653003201265100002101297700002201318700002001340700001901360856012401379 2006 eng d a0141-022900aStructural and active site modification studies implicate Glu, Trp and Arg in the activity of xylanase from alkalophilic Bacillus sp (NCL 87-6-10)0 aStructural and active site modification studies implicate Glu Tr a360 PARK AVE SOUTH, NEW YORK, NY 10010-1710 USAbELSEVIER SCIENCE INCcJUN a67-730 v393 a

Structural studies and residue modification using group specific reagents were used to ascertain the role of different functional groups in xylanase from alkalophilic Bacillus sp. (NCL 87-6-10). Treatment with N-bromosuccinimide resulted in fast enzyme inactivation. Reaction with Woodward's reagent K resulted in initial fast followed by slower inactivation. In both cases enzyme was protected against inactivation by the substrate, xylan. The reaction of the enzyme with phenylglyoxal has revealed one essential arginine residue at the active site. The three-dimensional structural analysis of the xylanase at 2.8 angstrom resolution also implicates involvement of Trp, and Arg residues and carboxylate groups in the binding of substrate and in the catalysis of xylanase. (c) 2005 Published by Elsevier Inc.

10aactive-site residues10aalkaline xylanase10aChemical modification10athree-dimensional structure1 aBalakrishnan, H.1 aSatyanarayana, L.1 aGaikwad, S., M.1 aSuresh, C., G. uhttps://library.ncl.res.in/content/structural-and-active-site-modification-studies-implicate-glu-trp-and-arg-activity-001980nas a2200217 4500008004100000022001400041245016600055210006900221260010300290300001600393490000800409520106400417100001801481700002501499700002101524700002401545700002001569700002401589700001901613856013001632 2006 eng d a0021-925800aStructural and functional analysis of a conjugated bile salt hydrolase from Bifidobacterium longum reveals an evolutionary relationship with penicillin V acylase0 aStructural and functional analysis of a conjugated bile salt hyd a9650 ROCKVILLE PIKE, BETHESDA, MD 20814-3996 USAbAMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INCcOCT a32516-325250 v2813 a

Bile salt hydrolase (BSH) is an enzyme produced by the intestinal microflora that catalyzes the deconjugation of glycine- or taurine-linked bile salts. The crystal structure of BSH reported here from Bifidobacterium longum reveals that it is a member of N-terminal nucleophil hydrolase structural superfamily possessing the characteristic alpha beta beta alpha tetra-lamellar tertiary structure arrangement. Site-directed mutagenesis of the catalytic nucleophil residue, however, shows that it has no role in zymogen processing into its corresponding active form. Substrate specificity was studied using Michaelis-Menten and inhibition kinetics and fluorescence spectroscopy. These data were compared with the specificity profile of BSH from Clostridium perfrigens and pencillin V acylase from Bacillus sphaericus, for both of which the three-dimensional structures are available. Comparative analysis shows a gradation in activity toward common substrates, throwing light on a possible common route toward the evolution of pencillin V acylase and BSH.

1 aKumar, Suresh1 aBrannigan, James, A.1 aPrabhune, Asmita1 aPundle, Archana, V.1 aDodson, Guy, G.1 aDodson, Eleanor, J.1 aSuresh, C., G. uhttps://library.ncl.res.in/content/structural-and-functional-analysis-conjugated-bile-salt-hydrolase-bifidobacterium-longum-003062nas a2200241 4500008004100000022001400041245011700055210006900172260008500241300000900326490000600335520215300341100002002494700002302514700002402537700002402561700002102585700001902606700002002625700002302645700002402668856012802692 2007 eng d a1932-620300aAnalysis of mitochondrial DNA sequences in childhood encephalomyopathies reveals new disease-associated variants0 aAnalysis of mitochondrial DNA sequences in childhood encephalomy a185 BERRY ST, STE 1300, SAN FRANCISCO, CA 94107 USAbPUBLIC LIBRARY SCIENCEcSEP ae9420 v23 a

Background. Mitochondrial encephalomyopathies are a heterogeneous group of clinical disorders generally caused due to mutations in either mitochondrial DNA (mtDNA) or nuclear genes encoding oxidative phosphorylation (OXPHOS). We analyzed the mtDNA sequences from a group of 23 pediatric patients with clinical and morphological features of mitochondrial encephalopathies and tried to establish a relationship of identified variants with the disease. Methodology/Principle Findings. Complete mitochondrial genomes were amplified by PCR and sequenced by automated DNA sequencing. Sequencing data was analyzed by SeqScape software and also confirmed by BLASTn program. Nucleotide sequences were compared with the revised Cambridge reference sequence (CRS) and sequences present in mitochondrial databases. The data obtained shows that a number of known and novel mtDNA variants were associated with the disease. Most of the non-synonymous variants were heteroplasmic (A4136G, A9194G and T11916A) suggesting their possibility of being pathogenic in nature. Some of the missense variants although homoplasmic were showing changes in highly conserved amino acids (T3394C, T3866C, and G9804A) and were previously identified with diseased conditions. Similarly, two other variants found in tRNA genes (G5783A and C8309T) could alter the secondary structure of Cys-tRNA and Lys-tRNA. Most of the variants occurred in single cases; however, a few occurred in more than one case (e. g. G5783A and A10149T). Conclusions and Significance. The mtDNA variants identified in this study could be the possible cause of mitochondrial encephalomyopathies with childhood onset in the patient group. Our study further strengthens the pathogenic score of known variants previously reported as provisionally pathogenic in mitochondrial diseases. The novel variants found in the present study can be potential candidates for further investigations to establish the relationship between their incidence and role in expressing the disease phenotype. This study will be useful in genetic diagnosis and counseling of mitochondrial diseases in India as well as worldwide.

1 aWani, Aijaz, A.1 aAhanger, Sajad, H.1 aBapat, Sharmila, A.1 aRangrez, Ashraf, Y.1 aHingankar, Nitin1 aSuresh, C., G.1 aBarnabas, Shama1 aPatole, Milind, S.1 aShouche, Yogesh, S. uhttps://library.ncl.res.in/content/analysis-mitochondrial-dna-sequences-childhood-encephalomyopathies-reveals-new-disease-002107nas a2200241 4500008004100000022001400041245014100055210006900196260008600265300001200351490000700363520118300370653001601553653002401569653001901593653001401612100001801626700001901644700002501663700002001688700002501708856013201733 2007 eng d a1521-654300aBile salt hydrolase, the member of Ntn-hydrolase family: differential modes of structural and functional transitions during denaturation0 aBile salt hydrolase the member of Ntnhydrolase family differenti a325 CHESTNUT ST, SUITE 800, PHILADELPHIA, PA 19106 USAbTAYLOR & FRANCIS INCcFEB a118-1250 v593 a

Conformational transitions and functional stability of the bile salt hydrolase (BSH; cholylglycine EC: 3.5.1.24) from Bifidobacterium longum (BlBSH) cloned and expressed in E. coli were studied under thermal, chemical and pH-mediated denaturation conditions using fluorescence and CD spectroscopy. Thermal and Gdn-HCl-mediated denaturation of BlBSH is a multistep process of inactivation and unfolding. The inactivation and unfolding of the enzyme was found to be irreversible. Enzyme activity seems sensitive to even minor conformational changes at the active site. Thermal denaturation as such did not result in any insoluble protein aggregates. However, on treating with 0.25-1 M Gdn-HCl the enzyme showed increasing aggregation at temperatures of 40-55 degrees C indicating more complex structural changes taking place in the presence of chemical denaturants. The enzyme secondary structure was still intact at acidic pH (pH 1-3). The perturbation in the tertiary structure at the acidic pH was detected through freshly formed solvent exposed hydrophobic patches on the enzyme. These changes could be due to the formation of an acid-induced molten globule-like state.

10aAggregation10aBile salt hydrolase10amolten-globule10aUnfolding1 aKumar, Suresh1 aSuresh, C., G.1 aBrannigan, James, A.1 aDodson, Guy, G.1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/bile-salt-hydrolase-member-ntn-hydrolase-family-differential-modes-structural-and-functional02062nas a2200205 4500008004100000022001400041245011100055210006900166260007300235300001400308490000900322520124800331653003501579653002901614653003201643653002201675100001601697700001901713856012401732 2007 eng d a1570-963900aBowman-birk protease inhibitor from the seeds of vigna unguiculata forms a highly stable dimeric structure0 aBowmanbirk protease inhibitor from the seeds of vigna unguiculat aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcOCT a1264-12730 v17743 a

Different protease inhibitors including Bowman-Birk type (BBI) have been reported from the seeds of rigna unguiculata. Protease isoinhibitors of double-headed Bowman-Birk type from the seeds of Vigna unguiculata have been purified and characterized. The BBI from Vigna unguiculata (Vu-BBI) has been found to undergo self-association to form very stable dimers and more complex oligomers, by size-exclusion chromatography and SDS-PAGE in the presence of urea. Many BBIs have been reported to undergo self-association to form homodimers or more complex oligomers in solution. Only one dimeric crystal structure of a BBI (pea-BBI) is reported to date. We report the three-dimensional structure of a Vu-BBI determined at 2.5 A resolution. Although, the inhibitor has a monomer fold similar to that found in other known structures of Bowman-Birk protease inhibitors, its quaternary structure is different from that commonly observed in this family. The structural elements responsible for the stability of monomer molecule and dimeric association are discussed. The Vu-BBI may use dimeric or higher quaternary association to maintain the physiological state and to execute its biological function. (C) 2007 Elsevier B.V. All rights reserved.

10aBowman-Birk protease inhibitor10aplant protease inhibitor10aprotein-protein interaction10aVigna unguiculata1 aRao, K., N.1 aSuresh, C., G. uhttps://library.ncl.res.in/content/bowman-birk-protease-inhibitor-seeds-vigna-unguiculata-forms-highly-stable-dimeric-002563nas a2200265 4500008004100000022001400041245011100055210006900166260007900235300001400314490000700328520157900335653002901914653001801943653002501961653002301986653002202009653002802031100001802059700002102077700001902098700003302117700001902150856012802169 2007 eng d a0141-022900aTryptophan residue is identified in the substrate binding of penicillin G acylase from Kluyvera citrophila0 aTryptophan residue is identified in the substrate binding of pen a360 PARK AVE SOUTH, NEW YORK, NY 10010-1710 USAbELSEVIER SCIENCE INCcAPR a1389-13970 v403 a

Penicillin acylases are important enzymes in pharmaceutical industry for the production of semi-synthetic beta-lactam antibiotics via the key intermediate 6-aminopenicillanic acid. The penicillin G acylase purified from Kluyvera citrophila (KcPGA) on modification with tryptophan-specific reagents such as N-bromo succinamide (NBS) and 2-hydroxy 5-nitrobenzylbromide (HNBB) showed partial loss of activity and substrate protection. Various solute quenchers and substrate were used to probe the microenvironment of the putative reactive tryptophan through fluorescence quenching. Homology modeling of KcPGA structure has been carried out. Docking substrate on this modeled KcPGA structure identifies the tryptophan residue that is directly influenced by substrate binding. To confirm the biological significance of this particular tryptophan, we did a sequence comparison of PGAs from various organisms. The sequence alignment clustered the matches into two sets, those closer to (> 40% identical) KcPGA and had the tryptophan of interest present in them formed the first set, while those less identical (< 30%) to KcPGA and the particular tryptophan absent in them formed the second set. It is clear from the reported kinetic parameters of representative members of these two sets that the affinity for penicillin G (penG) of the former class is several times better. Thus, based on our studies we suggest that the tryptophan residue in the identified position is important for binding substrate penG by the acylases. (c) 2006 Elsevier Inc. All rights reserved.

10afluorescence measurement10aK. citrophila10aPenicillin G acylase10asequence alignment10asubstrate-docking10atryptophan modification1 aKumar, Suresh1 aPrabhune, Asmita1 aPundle, A., V.1 aKarthikeyan, Muthukumarasamy1 aSuresh, C., G. uhttps://library.ncl.res.in/content/tryptophan-residue-identified-substrate-binding-penicillin-g-acylase-kluyvera-citrophila02357nas a2200277 4500008004100000022001400041245015900055210006900214260009000283300001200373490000700385520127400392653002401666653002801690653002901718653003401747100002001781700002401801700002001825700002401845700001901869700002001888700002301908700002001931856012801951 2008 eng d a1071-576200aAnalysis of reactive oxygen species and antioxidant defenses in complex I deficient patients revealed a specific increase in superoxide dismutase activity0 aAnalysis of reactive oxygen species and antioxidant defenses in  aTELEPHONE HOUSE, 69-77 PAUL STREET, LONDON EC2A 4LQ, ENGLANDbINFORMA HEALTHCAREcMAY a415-4270 v423 a

The mechanism of free radical production by complex I deficiency is ill-defined, although it is of significant contemporary interest. This study studied the ROS production and antioxidant defenses in children with mitochondrial NADH dehydrogenase deficiency. ROS production has remained significantly elevated in patients compared to controls. The expression of all antioxidant enzymes significantly increased at mRNA level. However, the enzyme activities did not correlate with high mRNA or protein expression. Only the activity of superoxide dismutase (SOD) was found to correlate with higher mRNA expression in patient derived cell lines. The activities of the enzymes such as glutathione peroxidase (GPx), Catalase (CAT) and glutathione-S-transferase (GST) were significantly reduced in patients (p < 0.05 or p < 0.01). Glutathione reductase (GR) activity and intracellular glutathione (GSH) levels were not changed. Decreased enzyme activities could be due to post-translational or oxidative modification of ROS scavenging enzymes. The information on the status of ROS and marking the alteration of ROS scavenging enzymes in peripheral lymphocytes or lymphoblast cell lines will provide a better way to design antioxidant therapies for such disorders.

10aantioxidant enzymes10amitochondrial complex I10amitochondrial myopathies10aReactive Oxygen Species (ROS)1 aWani, Aijaz, A.1 aRangrez, Ashraf, Y.1 aKumar, Himanshu1 aBapat, Sharmila, A.1 aSuresh, C., G.1 aBarnabas, Shama1 aPatole, Milind, S.1 aShouche, Yogesh uhttps://library.ncl.res.in/content/analysis-reactive-oxygen-species-and-antioxidant-defenses-complex-i-deficient-patients-002327nas a2200253 4500008004100000022001400041245013200055210006900187260009900256300001200355490000700367520135500374653002501729653002601754653002501780653001701805653002701822653002601849100001601875700002101891700001901912700002001931856012201951 2008 eng d a1359-511300aCharacterization of smallest active monomeric penicillin V acylase from new source: a yeast, rhodotorula aurantiaca (NCIM 3425)0 aCharacterization of smallest active monomeric penicillin V acyla aTHE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLANDbELSEVIER SCI LTDcSEP a961-9670 v433 a

An intracellular monomeric penicillin V acylase (PVA) of 36,000 Da exhibiting pI of 4.19, purified from newly identified yeast source, Rhodotorula aurantiaca (NCIM 3425). The enzyme was purified by hydrophobic interaction chromatography. The enzyme showed optimal activity at 45 degrees C and retained 80% activity after incubation at 45 degrees C and pH 5.5 for I h. The enzyme showed maximum activity at pH 5.5 and was very stable between pH 5.5-6.5 with optimum stability at pH 6.0. It exhibited 50% of its original activity after 30 min incubation at 60 degrees C. Enzyme hydrolyzed substrates with benzyl side chain but preferred penicillin V as primary substrate. N-terminally located serine supports the fact that it belongs to Ntn (N-terminal nucleophile)-hydrolase superfamily. The initial ten amino acid residues of R. aurantiaca PVA were identical to the initial sequence of NADH dehydrogenase (EC 1.6.99.3): however the enzyme lacks dehydrogenase activity. EGTA, EDTA, hexane and ethyl acetate stabilized the activity where as small chain alcohols inhibited it. 1,4-Dioxane, THF (tetrahydrofurane), phenol and benzyl alcohol severely inhibited enzyme activity while BME and DTT increased it. Tween 80 and Tween 20 highly enhanced the activity where as SIDS and Triton X-100 inhibited it. (C) 2008 Elsevier Ltd. All rights reserved.

10aIntracellular enzyme10aN-terminal sequencing10aPenicillin V acylase10aPurification10aRhodotorula aurantiaca10aSubstrate specificity1 aKumar, Atul1 aPrabhune, Asmita1 aSuresh, C., G.1 aPundle, Archana uhttps://library.ncl.res.in/content/characterization-smallest-active-monomeric-penicillin-v-acylase-new-source-yeast-002046nas a2200265 4500008004100000022001400041245009700055210006900152260007500221300001200296490000700308520112300315653001601438653001701454653001801471653001301489653002101502653002301523653002101546100001901567700001901586700002601605700002501631856012401656 2008 eng d a1053-050900aSteady state and time-resolved fluorescence studies of a hemagglutinin from Moringa oleifera0 aSteady state and timeresolved fluorescence studies of a hemagglu a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGER/PLENUM PUBLISHERScMAR a479-4850 v183 a

The saccharide binding and conformational characterization of a hemagglutinin, a low molecular weight protein from the seeds of Moringa oleifera was studied using steady state and time resolved fluorescence. The lectin binds sugars LacNAc (K-a = 1380 M-1) and fructose (K-a = 975 M-1), as determined by the fluorescence spectroscopy. It has a single tryptophan per monomer which is exposed on the surface and is in a strong electropositive environment as revealed by quenching with iodide. Quenching of the fluorescence by acrylamide involved both static (K-s = 0.216 M-1) and collisional (K-sv= 8.19 M-1) components. The native protein showed two different lifetimes, tau(1) (1.6 ns) and tau(2) (4.36 ns) which decrease and get converted into a single one, (2.21 ns) after quenching with 0.15 M acrylamide. The bimolecular quenching constant, k(q) was 7.55 x 10(11) M-1 s(-1). ANS binding studies showed that the native protein has exposed hydrophobic patches which get further exposed at extreme acidic or alkaline pH. However, they get buried in the interior of the protein in presence of 1 M GdnHCl or urea.

10aANS binding10afluorescence10aHemagglutinin10alifetime10aMoringa oleifera10asaccharide binding10aSolute quenching1 aKatre, Uma, V.1 aSuresh, C., G.1 aKhan, Mohammad, Islam1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/steady-state-and-time-resolved-fluorescence-studies-hemagglutinin-moringa-oleifera-001652nas a2200217 4500008004100000022001400041245008300055210006900138260007300207300001200280490000700292520085400299653002301153653003501176653002401211100001901235700001901254700002601273700002501299856011001324 2008 eng d a0141-813000aStructure-activity relationship of a hemagglutinin from Moringa oleifera seeds0 aStructureactivity relationship of a hemagglutinin from Moringa o aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcMAR a203-2070 v423 a

The hemagglutinin from the seeds of Moringa oleifera (MoL) agglutinates human as well as rabbit erythrocytes; the affinity for the latter is almost 250 times more than that for the former. MoL was inhibited by glycoproteins namely thyroglobulin, fetuin and holotransferin indicating the complex sugar specificity of the lectin. The protein is a homodimer with molecular mass of 14 kDa, subunits (7.1 kDa) linked by the disulfide bond(s). The secondary structure elements of MoL area-helix, 28%; beta-sheet, 23%; turn 20% and unordered 28%. While the activity and secondary structure were not affected at extreme pH and high temperature, they were drastically affected in presence of dithiothreitol at and above pH 7.0, indicating that disulfide linkages hold the active conformation of the protein. (C) 2007 Elsevier B.V. All rights reserved.

10adisulfide linkages10aMoringa oleifera hemagglutinin10aSecondary structure1 aKatre, Uma, V.1 aSuresh, C., G.1 aKhan, Mohammad, Islam1 aGaikwad, Sushama, M. uhttps://library.ncl.res.in/content/structure-activity-relationship-hemagglutinin-moringa-oleifera-seeds-002108nas a2200277 4500008004100000022001400041245011500055210006900170260007500239300001200314490000700326520110400333653002001437653001701457653001701474653002601491653002401517100002401541700002501565700001901590700001801609700002601627700002001653700002701673856013001700 2009 eng d a1053-050900aComparative studies of two araceous lectins by steady state and time-resolved fluorescence and CD spectroscopy0 aComparative studies of two araceous lectins by steady state and  a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGER/PLENUM PUBLISHERScMAR a239-2480 v193 a

Transitions in the tryptophan microenvironment and secondary structure of two monocot lectins from Sauromatum guttatum and Arisaema tortuosum under different denaturing conditions were studied by steady state and time resolved fluorescence and CD spectroscopy. The lectins exist as tetramers with a single tryptophan residue estimated per monomer, present in a polar environment. Quenching with ionic quenchers showed predominantly electropositive environment for tryptophan residues. Acrylamide had maximum quenching effect. A decrease in KI quenching due to lectin denaturation indicated redistribution of charges as a result of possible conformational change. The two values for lifetimes of tryptophanyl population (1.2-1.4 and 6.3-6.4 ns) reduced substantially on quenching or denaturation. Similarly, both the lectins showed a drastic loss of secondary structure in 5 M Gdn-HCl or 6 M Urea or at pH 2.0 and below. For the first time araceous lectins, like legume lectins are shown to bind adenine. The presence of a compact structure at alkaline pH 10.0-12.0 was observed in CD spectra.

10aAraceae lectins10aDenaturation10afluorescence10aLifetime spectroscopy10aSecondary structure1 aDharker, Poorva, N.1 aGaikwad, Sushama, M.1 aSuresh, C., G.1 aDhuna, Vikram1 aKhan, Mohammad, Islam1 aSingh, Jatinder1 aKamboj, Sukhdev, Singh uhttps://library.ncl.res.in/content/comparative-studies-two-araceous-lectins-steady-state-and-time-resolved-fluorescence-and-002426nas a2200277 4500008004100000022001400041245011100055210006900166260009500235300001200330490000800342520139600350653002301746653001601769653001501785653001901800653004801819100002101867700002401888700002601912700002201938700001901960700002501979700002102004856012302025 2009 eng d a0006-291X00aConformational characterization of human eukaryotic initiation factor 2 alpha: a single tryptophan protein0 aConformational characterization of human eukaryotic initiation f a525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USAbACADEMIC PRESS INC ELSEVIER SCIENCEcDEC a273-2790 v3903 a

The alpha-subunit of the human eukaryotic initiation factor 2 (heIF2 alpha), a GTP binding protein, plays a major role in the initiation of protein synthesis. During various cytoplasmic stresses, eIF2 alpha gets phosphorylated by eIF2 alpha-specific kinases resulting in inhibition of protein synthesis. The cloned and over expressed heIF2 alpha, a protein with a single tryptophan (trp) residue was examined for its conformational characteristics using steady-state and time-resolved tryptophan fluorescence, circular dichroism (CD) and hydrophobic dye binding. The steady-state fluorescence spectrum, fluorescence lifetimes (tau(1) = 1.13 ns and tau(2) = 4.74 ns) and solute quenching studies revealed the presence of trp conformers in hydrophobic and differential polar environment at any given time. Estimation of the alpha-helix and beta-sheet content showed: (i) more compact structure at pH 2.0, (ii) distorted alpha-helix and rearranged beta-sheet in presence of 4 M guanidine hydrochloride and (iii) retention of more than 50% ordered structure at 95 degrees C. Hydrophobic dye binding to the protein with loosened tertiary structure was observed at pH 2.0 indicating the existence of a molten globule-like structure. These observations indicate the inherent structural stability of the protein under various denaturing conditions. (C) 2009 Elsevier Inc. All rights reserved.

10aCircular dichroism10aheIF2 alpha10aK562 cells10aMolten globule10aSteady-state and time-resolved fluorescence1 aSreejith, R., K.1 aYadav, Viveka, Nand1 aVarshney, Nishant, K.1 aBerwal, Sunil, K.1 aSuresh, C., G.1 aGaikwad, Sushama, M.1 aPal, Jayanta, K. uhttps://library.ncl.res.in/content/conformational-characterization-human-eukaryotic-initiation-factor-2-alpha-single-001916nas a2200157 4500008004100000022001400041245007300055210006900128260009200197300000900289490000700298520131100305100001901616700001901635856010401654 2009 eng d a0907-444900aFeatures of homotetrameric molecular association in protein crystals0 aFeatures of homotetrameric molecular association in protein crys aCOMMERCE PLACE, 350 MAIN ST, MALDEN 02148, MA USAbWILEY-BLACKWELL PUBLISHING, INCcJAN a1-100 v653 a

The crystal structures of proteins showing homotetrameric association, a common feature observed in many lectins, have been analyzed in order to understand the characteristics of tetrameric association in terms of the arrangement of subunits and their biological significance. The analysis could group the tetramer units into the following four categories. (i) Tetrahedral molecules, in which the four monomers form a nearly perfect tetrahedral arrangement. The angle between the axes of any two monomers is similar to 109 degrees. (ii) Molecules that form a sandwiched dimer of dimers in which the two dimers are arranged perpendicular to each other, one upon the other. (iii) Planar molecules, in which the four monomers lie in one plane and the corresponding sides of adjacent monomers face in opposite directions. This can be considered as a flattened tetrahedral shape. (iv) Planar closed molecules, in which all four monomers lie in one plane arranged in a head-to-tail fashion in a square. The first group and its variant, the third group, are the most commonly found arrangements in crystal structures. Each arrangement has its own importance for biological function. Some tetrameric assemblies that deviate from the majority described above also have relevance to their biological function.

1 aKatre, Uma, V.1 aSuresh, C., G. uhttps://library.ncl.res.in/content/features-homotetrameric-molecular-association-protein-crystals-002160nas a2200289 4500008004100000022001400041245011900055210006900174260007500243300001200318490000700330520114000337653001201477653002601489653001801515653001901533653002401552653002001576653001401596100002001610700002501630700001901655700001801674700002001692700002701712856013101739 2011 eng d a1053-050900aConformational transitions in ariesaema curvatum lectin: characterization of an acid induced active molten globule0 aConformational transitions in ariesaema curvatum lectin characte a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGER/PLENUM PUBLISHERScMAR a753-7630 v213 a

Biophysical characterization of a lectin from Ariesaema curvatum (ACL) was carried out using steady state as well as time resolved fluorescence and CD spectroscopy under various denaturing conditions. An intermediate with altered tryptophan microenvironment was detected in the phase diagram, which exibited pronounced secondary structure and hemagglutinating activity in presence of 0.25 M Gdn-HCl. An acid induced molten- globule like structure possessing activity and higher thermostability was detected. Transition to the molten globule state was reversible in nature. The lectin retained hemagglutinating activity even after incubation at 95 A degrees C. Both chemical and thermal unfolding of the lectin were found to consist of multistate processes. Fluorescence quenching of ACL was strong with acrylamide and KI. The single tryptophan was found to be surrounded by high density of the positively charged amino acid residues as shown by a ten fold higher K(sv) for KI compared to that for CsCl. The average lifetime of tryptophan fluorescence increased from 1.24 ns in the native state to 1.72 ns in the denatured state.

10aAraceae10aFluorescence lifetime10aHemagglutinin10aMolten globule10aSecondary structure10aThermostability10aUnfolding1 aSharma, Urvashi1 aGaikwad, Sushama, M.1 aSuresh, C., G.1 aDhuna, Vikram1 aSingh, Jatinder1 aKamboj, Sukhdev, Singh uhttps://library.ncl.res.in/content/conformational-transitions-ariesaema-curvatum-lectin-characterization-acid-induced-active-001624nas a2200193 4500008004100000022001400041245012700055210006900182260007600251300001400327490000700341520084900348653002001197653002001217100002001237700001901257700002401276856013001300 2011 eng d a1744-309100aPurification, crystallization and preliminary X-ray characterization of a haemagglutinin from the seeds of Jatropha curcas0 aPurification crystallization and preliminary Xray characterizati aCOMMERCE PLACE, 350 MAIN ST, MALDEN 02148, MA USAbWILEY-BLACKWELLcDEC a1534-15360 v673 a

The plant Jatropha curcas (Euphorbiaceae) is an important source of biofuel from the inedible oil present in its toxic seeds. The toxicity arises from the presence of curcin, a ribosome-inactivating protein showing haemagglutination activity. In this communication, the purification, crystallization and preliminary X-ray characterization are reported of a small protein isolated from J. curcas seeds with a molecular mass of 10 kDa that agglutinates rabbit erythrocytes. The protein was crystallized using the hanging-drop vapour-diffusion method and also by the microbatch method in 72-well HLA plates, using PEG 8000 as the precipitant in both conditions. X-ray diffraction data collected from the rod-shaped crystals were processed in the orthorhombic space group P212121. The crystals diffracted to 2.8 angstrom resolution at 103 K.

10ahaemagglutinins10aJatropha curcas1 aNair, Divya, N.1 aSuresh, C., G.1 aSingh, Desh, Deepak uhttps://library.ncl.res.in/content/purification-crystallization-and-preliminary-x-ray-characterization-haemagglutinin-seeds-001291nas a2200157 4500008004100000022001400041245014900055210006900204260006700273300001200340490000700352520060800359100002000967700001900987856012701006 2011 eng d a1744-309100aPurification, crystallization and X-ray characterization of a Kunitz-type trypsin inhibitor protein from the seeds of chickpea (Cicer arietinum)0 aPurification crystallization and Xray characterization of a Kuni a111 RIVER ST, HOBOKEN 07030-5774, NJ USAbWILEY-BLACKWELLcJUN a714-7170 v673 a

{A Kunitz-type trypsin inhibitor protein (CPTI) purified from chickpea seeds was estimated to have a molecular mass of 18 kDa on SDS-PAGE. The IC50 value of CPTI was determined to be 2.5 mu g against trypsin. The inhibitory activity of CPTI is 114 TIU (trypsin inhibitory units) per milligram of protein, which is high compared with those of other known Kunitz-type trypsin inhibitors from legumes. CPTI crystallized in three different orthorhombic crystal forms: P2(1)2(1)2 form A, P2(1)2(1)2 form B and P2(1)2(1)2(1). The crystals of P2(1)2(1)2 form angstrom, with unit-cell parameters a = 37.2

1 aSharma, Urvashi1 aSuresh, C., G. uhttps://library.ncl.res.in/content/purification-crystallization-and-x-ray-characterization-kunitz-type-trypsin-inhibitor-002631nas a2200277 4500008004100000022001400041245015800055210006900213260007500282300001200357490000700369520154700376653002801923653002201951653002901973653001902002653004802021100002102069700001902090700002702109700002102136700001902157700002502176700002102201856013102222 2012 eng d a1053-050900aConformational transitions of the catalytic domain of heme-regulated eukaryotic initiation factor 2 alpha kinase, a key translational regulatory molecule0 aConformational transitions of the catalytic domain of hemeregula a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGER/PLENUM PUBLISHERScJAN a431-4410 v223 a

In mammalian cells, the heme-regulated inhibitor (HRI) plays a critical role in the regulation of protein synthesis at the initiation step through phosphorylation of a-subunit of the eukaryotic initiation factor 2 (eIF2). In this study we have cloned and performed biophysical characterization of the kinase catalytic domain (KD) of rabbit HRI. The KD described here comprises kinase 1, the kinase insertion domain (KI) and kinase 2. We report here the existence of an active and stable monomer of HRI (KD). The HRI (KD) containing three tryptophan residues was examined for its conformational transitions occurring under various denaturing conditions using steady-state and time-resolved tryptophan fluorescence, circular dichroism (CD) and hydrophobic dye binding. The parameter A and phase diagram analysis revealed multi-state unfolding and existence of three stable intermediates during guanidine hydrochloride (Gdn-HCl) induced unfolding of HRI (KD). The protein treated with 6 M Gdn-HCl showed collisional and static mechanism of acrylamide quenching and the constants (K-sv=3.08 M-1 and K-s=5.62 M-1) were resolved using time resolved fluorescence titration. Based on pH, guanidine hydrochloride and temperature mediated transitions, HRI (KD) appears to exemplify a rigid molten globule-like intermediate with compact secondary structure, altered tertiary structure and exposed hydrophobic patches at pH 3.0. The results indicate the inherent structural stability of HRI (KD), a member of the class of stress response proteins.

10aCircular dichroism (CD)10aeIF2 alpha kinase10aHeme-regulated inhibitor10aMolten globule10aSteady-state and time-resolved fluorescence1 aSreejith, R., K.1 aSuresh, C., G.1 aBhosale, Siddharth, H.1 aBhavnani, Varsha1 aKumar, Avinash1 aGaikwad, Sushama, M.1 aPal, Jayanta, K. uhttps://library.ncl.res.in/content/conformational-transitions-catalytic-domain-heme-regulated-eukaryotic-initiation-factor-2-001476nas a2200289 4500008004100000022001400041245011400055210006900169260007600238300001200314490000700326520044800333653002000781653002200801653001900823653002200842653002000864653002000884100002900904700001800933700001900951700002100970700002000991700002401011700001901035856013201054 2012 eng d a1744-309100aCrystallization and X-ray structure analysis of a thermostable penicillin G acylase from alcaligenes faecalis0 aCrystallization and Xray structure analysis of a thermostable pe aCOMMERCE PLACE, 350 MAIN ST, MALDEN 02148, MA USAbWILEY-BLACKWELLcMAR a273-2770 v683 a

{The enzyme penicillin G acylase (EC 3.5.1.11) catalyzes amide-bond cleavage in benzylpenicillin (penicillin G) to yield 6-aminopenicillanic acid, an intermediate chemical used in the production of semisynthetic penicillins. A thermostable penicillin G acylase from Alcaligenes faecalis (AfPGA) has been crystallized using the hanging-drop vapour-diffusion method in two different space groups: C2221, with unit-cell parameters a = 72.9

10acalcium binding10adisulfide bridges10aNtn hydrolases10aorthorhombic form10atetragonal form10aThermostability1 aVarshney, Nishant, Kumar1 aKumar, Suresh1 aIgnatova, Zoya1 aPrabhune, Asmita1 aPundle, Archana1 aDodson, Eleanor, J.1 aSuresh, C., G. uhttps://library.ncl.res.in/content/crystallization-and-x-ray-structure-analysis-thermostable-penicillin-g-acylase-alcaligenes-002185nas a2200265 4500008004100000022001400041245009100055210006900146260007300215300001000288490000700298520124000305653002201545653003601567653001801603653002301621100002401644700002501668700001901693700002101712700002501733700002001758700002401778856011701802 2012 eng d a0141-813000aPurification and characterization of YxeI, a penicillin acylase from Bacillus subtilis0 aPurification and characterization of YxeI a penicillin acylase f aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcJAN a25-300 v503 a

The paper reports the purification and characterization of the first penicillin acylase from Bacillus subtilis. YxeI, the protein annotated as hypothetical, coded by the gene yxeI in the open reading frame between iol and hut operons in B. subtilis was cloned and expressed in Eshcherichia coli, purified and characterized. The purified protein showed measurable penicillin acylase activity with penicillin V. The enzyme was a homotetramer of 148 kDa. The apparent Km of the enzyme for penicillin V and the synthetic substrate 2-nitro-5-(phenoxyacetamido)-benzoic acid was 40 mM and 0.63 mM, respectively, and the association constants were 8.93 x 10(2) M-1 and 2.51 x 10(5) M-1, respectively. It was inhibited by cephalosporins and conjugated bile salts, substrates of the closely related bile acid hydrolases. It had good sequence homology with other penicillin V acylases and conjugated bile acid hydrolases, members of the Ntn hydrolase family. The N-terminal nucleophile was a cysteine which is revealed by a simple removal of N-formyl-methionine. The activity of the protein was affected by high temperature, acidic pH and the presence of the denaturant guanidine hydrochloride. (C) 2011 Elsevier B.V. All rights reserved.

10aBacillus subtilis10aConjugated bile acid hydrolases10aNtn hydrolase10aPenicillin acylase1 aRathinaswamy, Priya1 aGaikwad, Sushama, M.1 aSuresh, C., G.1 aPrabhune, Asmita1 aBrannigan, James, A.1 aDodson, Guy, G.1 aPundle, Archana, V. uhttps://library.ncl.res.in/content/purification-and-characterization-yxei-penicillin-acylase-bacillus-subtilis-001780nas a2200193 4500008004100000022001400041245017000055210006900225260006700294300001200361490000700373520095200380100002901332700002601361700002501387700002701412700001901439856012801458 2013 eng d a1744-309100aCloning, overexpression, crystallization and preliminary X-ray crystallographic analysis of a slow-processing mutant of penicillin G acylase from Kluyvera citrophila0 aCloning overexpression crystallization and preliminary Xray crys a111 RIVER ST, HOBOKEN 07030-5774, NJ USAbWILEY-BLACKWELLcAUG a925-9290 v693 a

{Kluyvera citrophila penicillin G acylase (KcPGA) has recently attracted increased attention relative to the well studied and commonly used Escherichia coli PGA (EcPGA) because KcPGA is more resilient to harsh conditions and is easier to immobilize for the industrial hydrolysis of natural penicillins to generate the 6-aminopenicillin (6-APA) nucleus, which is the starting material for semi-synthetic antibiotic production. Like other penicillin acylases, KcPGA is synthesized as a single-chain inactive pro-PGA, which upon autocatalytic processing becomes an active heterodimer of alpha and beta chains. Here, the cloning of the pac gene encoding KcPGA and the preparation of a slow-processing mutant precursor are reported. The purification, crystallization and preliminary X-ray analysis of crystals of this precursor protein are described. The protein crystallized in two different space groups, P1, with unit-cell parameters a = 54.0

1 aVarshney, Nishant, Kumar1 aRamasamy, Sureshkumar1 aBrannigan, James, A.1 aWilkinson, Anthony, J.1 aSuresh, C., G. uhttps://library.ncl.res.in/content/cloning-overexpression-crystallization-and-preliminary-x-ray-crystallographic-analysis-002516nas a2200265 4500008004100000022001400041245012500055210006900180260007300249300001400322490000900336520153000345653001901875653002101894653003601915653001301951653002301964653003201987100002202019700001902041700001902060700002102079700002002100856013002120 2013 eng d a0304-416500aComplementation of intramolecular interactions for structural-functional stability of plant serine proteinase inhibitors0 aComplementation of intramolecular interactions for structuralfun aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcNOV a5087-50940 v18303 a

Background: Plant protease inhibitors (PIs) constitute a diverse group of proteins capable of inhibiting proteases. Among Pls, serine Pls (SPIs) display stability and conformational restrictions of the reactive site loop by virtue of their compact size, and by the presence of disulfide bonds, hydrogen bonds, and other weak interactions. Scope of review: The significance of various intramolecular interactions contributing to protein folding mechanism and their role in overall stability and activity of SPIs is discussed here. Furthermore, we have reviewed the effect of variation or manipulation of these interactions on the activity/stability of SPIs. Major conclusions: The selective gain or loss of disulfide bond(s) in SPIs can be associated with their functional differentiation, which is likely to be compensated by non-covalent interactions (hydrogen bonding or electrostatic interactions). Thus, these intramolecular interactions are collectively responsible for the functional activity of SPIs, through the maintenance of scaffold framework, conformational rigidity and shape complementarities of reactive site loop. General significance: Structural insight of these interactions will provide an in-depth understanding of kinetic and thermodynamic parameters involved in the folding and stability mechanisms of SPIs. These features can be explored for engineering canonical SPIs for optimizing their overall stability and functionality for various applications. (C) 2013 Elsevier B.V. All rights reserved.

10aDisulfide bond10ahydrogen bonding10aIntramolecular weak interaction10aProtease10aReactive site loop10aSerine proteinase inhibitor1 aJoshi, Rakesh, S.1 aMishra, Manasi1 aSuresh, C., G.1 aGupta, Vidya, S.1 aGiri, Ashok, P. uhttps://library.ncl.res.in/content/complementation-intramolecular-interactions-structural-functional-stability-plant-serine-002087nas a2200229 4500008004100000022001400041245011300055210006900168260011100237300001200348490000700360520113600367653002501503653005401528653002501582653003201607100002201639700002501661700002001686700001901706856013201725 2013 eng d a0929-866500aCrystal structure and fluorescence analysis of alkaline thermostable xylanase from bacillus sp (NCL 87-6-10)0 aCrystal structure and fluorescence analysis of alkaline thermost aEXECUTIVE STE Y-2, PO BOX 7917, SAIF ZONE, 1200 BR SHARJAH, U ARAB EMIRATESbBENTHAM SCIENCE PUBL LTDcFEB a125-1320 v203 a

Structural information deduced from the new crystal form of xylanase from Bacillus sp (NCL 87-6-10) (ATBXYL-C) helped us to identify the active site and interpret the stability of the enzyme. The analysis of the tetragonal crystal structure of ATBXYL-C with a bound and cleaved xylotriose revealed the two glutamic acid residues in the structure that could act as nucleophile (Glu94) and base (Glu184) in the enzyme activity and also the tryptophan residues interacting with the substrate. The cleavage of xylotriose in the crystal showed xylobiose to be the major product. Intrinsic fluorescence of the enzyme showed the presence of tryptophans in partially exposed to the solvent at the active site and surface tryptophans in electropositive environment. The titration experiments with xylobiose and xylotriose revealed slightly enhanced preference for longer chain X3 compared with X2. The crystal structure also account for some of the factors, such as increased number of ionic interactions and additional interactions at the N-terminus, which contributed to increased alkalophilicity and thermostability of the enzyme.

10aactive site residues10aAlkaline thermophile Bacillus xylanase (ATBXYL-C)10afluorescence studies10athree-dimensional structure1 aSatyanarayana, L.1 aGaikwad, Sushama, M.1 aBalkrishnan, H.1 aSuresh, C., G. uhttps://library.ncl.res.in/content/crystal-structure-and-fluorescence-analysis-alkaline-thermostable-xylanase-bacillus-sp-ncl-002710nas a2200241 4500008004100000022001400041245011900055210006900174260010300243300001400346490000700360520177700367653003802144653001402182653001902196653001102215100002302226700002202249700002402271700002502295700001902320856012902339 2013 eng d a0300-908400aHuman mitochondrial NDUFS3 protein bearing leigh syndrome mutation is more prone to aggregation than its wild-type0 aHuman mitochondrial NDUFS3 protein bearing leigh syndrome mutati a23 RUE LINOIS, 75724 PARIS, FRANCEbELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIERcDEC a2392-24030 v953 a

NDUFS3 is an integral subunit of the Q module of the mitochondrial respiratory Complex-I. The combined mutation (T145I + R199W) in the subunit is reported to cause optic atrophy and Leigh syndrome accompanied by severe Complex-I deficiency. In the present study, we have cloned and overexpressed the human NDUFS3 subunit and its double mutant in a soluble form in Escherichia coli. The wild-type (w-t) and mutant proteins were purified to homogeneity through a serial two-step chromatographic purification procedure of anion exchange followed by size exclusion chromatography. The integrity and purity of the purified proteins was confirmed by Western blot analysis and MALDI-TOF/TOF. The conformational transitions of the purified subunits were studied through steady state as well as time resolved fluorescence and CD spectroscopy under various denaturing conditions. The mutant protein showed altered polarity around tryptophan residues, changed quenching parameters and also noticeably altered secondary and tertiary structure compared to the w-t protein. Mutant also exhibited a higher tendency than the w-t protein for aggregation which was examined using fluorescent (Thioflavin-T) and spectroscopic (Congo red) dye binding techniques. The pH stability of the w-t and mutant proteins varied at extreme acidic pH and the molten globule like structure of w-t at pH1 was absent in case of the mutant protein. Both the w-t and mutant proteins showed multi-step thermal and Gdn-HCI induced unfolding. Thus, the results provide insight into the alterations of NDUFS3 protein structure caused by the mutations, affecting the overall integrity of the protein and finally leading to disruption of Complex-I assembly. (C) 2013 Elsevier Masson SAS. All rights reserved.

10aAggregation Fluorescence lifetime10aComplex-I10aLeigh syndrome10aNDUFS31 aJaokar, Tulika, M.1 aPatil, Deepak, P.1 aShouche, Yogesh, S.1 aGaikwad, Sushama, M.1 aSuresh, C., G. uhttps://library.ncl.res.in/content/human-mitochondrial-ndufs3-protein-bearing-leigh-syndrome-mutation-more-prone-aggregation01922nas 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-000635nas a2200193 4500008004100000022001400041245007400055210006900129260000800198300000900206490000700215653001300222653002100235653001300256100002500269700002600294700001900320856010200339 2014 eng d a2053-273300aChickpea Kunitz Inhibitor: a mechanistic basis for trypsin inhibition0 aChickpea Kunitz Inhibitor a mechanistic basis for trypsin inhibi cMAR aC2710 v7010aChickpea10aKunitz Inhibitor10aProtease1 aBendre, Ameya, Dipak1 aRamasamy, Sureshkumar1 aSuresh, C., G. uhttps://library.ncl.res.in/content/chickpea-kunitz-inhibitor-mechanistic-basis-trypsin-inhibition01915nas a2200169 4500008004100000022001400041245016600055210006900221260009100290300001200381490000600393520118100399100001701580700001701597700001901614856011201633 2014 eng d a1932-620300aGenome-wide identification and tissue-specific expression analysis of UDP-glycosyltransferases genes confirm their abundance in cicer arietinum (chickpea) genome0 aGenomewide identification and tissuespecific expression analysis a1160 BATTERY STREET, STE 100, SAN FRANCISCO, CA 94111 USAbPUBLIC LIBRARY SCIENCEcOCT ae1097150 v93 a

UDP-glycosyltransferases (EC 2.4.1.x; UGTs) are enzymes coded by an important gene family of higher plants. They are involved in the modification of secondary metabolites, phytohormones, and xenobiotics by transfer of sugar moieties from an activated nucleotide molecule to a wide range of acceptors. This modification regulates various functions like detoxification of xenobiotics, hormone homeostasis, and biosynthesis of secondary metabolites. Here, we describe the identification of 96 UGT genes in Cicer arietinum (CaUGT) and report their tissue-specific differential expression based on publically available RNA-seq and expressed sequence tag data. This analysis has established medium to high expression of 84 CaUGTs and low expression of 12 CaUGTs. We identified several closely related orthologs of CaUGTs in other genomes and compared their exon-intron arrangement. An attempt was made to assign functional specificity to chickpea UGTs by comparing substrate binding sites with experimentally determined specificity. These findings will assist in precise selection of candidate genes for various applications and understanding functional genomics of chickpea.

1 aSharma, Ranu1 aRawat, Vimal1 aSuresh, C., G. uhttps://library.ncl.res.in/content/genome-wide-identification-and-tissue-specific-expression-analysis-udp-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-002993nas a2200289 4500008004100000022001400041245017700055210006900232260008600301300001000387490000700397520189300404653002002297653002502317653001402342653002502356100002202381700001902403700002802422700001902450700002402469700001902493700002002512700002102532700002002553856013002573 2014 eng d a0739-110200aRemarkable efficiency of a pin-II proteinase inhibitor sans two conserved disulfide bonds is due to enhanced flexibility and hydrogen bond density in the reactive site loop0 aRemarkable efficiency of a pinII proteinase inhibitor sans two c a325 CHESTNUT ST, SUITE 800, PHILADELPHIA, PA 19106 USAbTAYLOR & FRANCIS INCcJAN a13-260 v323 a

Capsicum annuum (L.) expresses diverse potato type II family proteinase inhibitors comprising of inhibitory repeat domain (IRD) as basic functional unit. Most IRDs contain eight conserved cysteines forming four disulfide bonds, which are indispensible for their stability and activity. We investigated the functional significance of evolutionary variations in IRDs and their role in mediating interaction between the inhibitor and cognate proteinase. Among the 18 IRDs encoded by C. annuum, IRD-7, -9, and -12 were selected for further characterization on the basis of variation in their reactive site loop, number of conserved cysteine residues, and higher theoretical G(bind) for interaction with Helicoverpa armigera trypsin. Moreover, inhibition kinetics showed that IRD-9, despite loss of some of the disulfide bonds, was a more potent proteinase inhibitor among the three selected IRDs. Molecular dynamic simulations revealed that serine residues in the place of cysteines at seventh and eighth positions of IRD-9 resulted in an increase in the density of intramolecular hydrogen bonds and reactive site loop flexibility. Results of the serine residues chemical modification also supported this observation and provided a possible explanation for the remarkable inhibitory potential of IRD-9. Furthermore, this natural variant among IRDs showed special attributes like stability to proteolysis and synergistic inhibitory effect on other IRDs. It is likely that IRDs have coevolved selective specialization of their structure and function as a response towards specific insect proteases they encountered. Understanding the molecular mechanism of pest protease-plant proteinaceous inhibitor interaction will help in developing effective pest control strategies.An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:39

10aCapsicum annuum10aHelicoverpa armigera10aProteases10aProteinase inhibitor1 aJoshi, Rakesh, S.1 aMishra, Manasi1 aTamhane, Vaijayanti, A.1 aGhosh, Anirban1 aSonavane, Uddhavesh1 aSuresh, C., G.1 aJoshi, Rajendra1 aGupta, Vidya, S.1 aGiri, Ashok, P. uhttps://library.ncl.res.in/content/remarkable-efficiency-pin-ii-proteinase-inhibitor-sans-two-conserved-disulfide-bonds-due-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-excision02756nas a2200241 4500008004100000022001400041245012900055210006900184260005700253300001400310490000800324520187300332653002402205653002602229653002002255653001902275653001602294653001802310100002002328700001902348700001902367856012802386 2015 eng d a0032-093500aCrystal structure of a plant albumin from cicer arietinum (chickpea) possessing hemopexin fold and hemagglutination activity0 aCrystal structure of a plant albumin from cicer arietinum chickp a233 SPRING ST, NEW YORK, NY 10013 USAbSPRINGERcMAY a1061-10730 v2413 a

Crystal structure of a reported PA2 albumin from Cicer arietinum shows that it belongs to hemopexin fold family, has four beta-propeller motifs and possesses hemagglutination activity, making it different from known legume lectins. A plant albumin (PA2) from Cicer arietinum, presumably a lectin (CAL) owing to its hemagglutination activity which is inhibited by complex sugars as well as glycoproteins such as fetuin, desialylated fetuin and fibrinogen. The three-dimensional structure of this homodimeric protein has been determined using X-ray crystallography at 2.2 in two crystal forms: orthorhombic (P2(1)2(1)2) and trigonal (P3). The structure determined using molecular replacement method and refined in orthorhombic crystal form reached R-factors R (free) 22.6 % and R (work) 18.2 % and in trigonal form had 22.3 and 17.9 % in the resolution range of 20.0-2.2 and 35.3-2.2 , respectively. Interestingly, unlike the known legume lectin fold, the structure of this homodimeric hemagglutinin belonged to hemopexin fold that consisted of four-bladed beta-propeller architecture. Each subunit has a central cavity forming a channel, inside of which is lined with hydrophobic residues. The channel also bears binding sites for ligands such as calcium, sodium and chloride ions, iodine atom in the case of iodine derivative and water molecules. However, none of these ligands seem important for the sugar recognition. No monosaccharide sugar specificity could be detected using hemagglutination inhibition. Chemical modification studies identified a potential sugar-binding site per subunit molecule. Comparison of C-alpha atom positions in subunit structures showed that the deviations between the two crystal forms were more with respect to blades I and IV. Differences also existed between subunits in two forms in terms of type and site of ligand binding.

10aBeta-propeller fold10aChemical modification10aChickpea lectin10aComplex sugars10aPA2 albumin10aSugar binding1 aSharma, Urvashi1 aKatre, Uma, V.1 aSuresh, C., G. uhttps://library.ncl.res.in/content/crystal-structure-plant-albumin-cicer-arietinum-chickpea-possessing-hemopexin-fold-and-002373nas 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-102809nas a2200265 4500008004100000022001400041245012900055210006900184260010600253300001000359490000700369520184100376653001102217653002002228653002002248653002302268653002202291653003402313653001002347653001402357653001402371100001702385700001902402856012202421 2015 eng d a0010-482500aGenome-wide identification and structure-function studies of proteases and protease inhibitors in cicer arietinum (chickpea)0 aGenomewide identification and structurefunction studies of prote aTHE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, ENGLANDbPERGAMON-ELSEVIER SCIENCE LTDcJAN a67-810 v563 a

Background: Proteases are a family of enzymes present in almost all living organisms. In plants they are involved in many biological processes requiring stress response in situations such as water deficiency, pathogen attack, maintaining protein content of the cell, programmed cell death, senescence, reproduction and many more. Similarly, protease inhibitors (PIs) are involved in various important functions like suppression of invasion by pathogenic nematodes, inhibition of spores-germination and mycelium growth of Alternaria alternata and response to wounding and fungal attack. As much as we know, no genome-wide study of proteases together with proteinaceous PIs is reported in any of the sequenced genomes till now. Methods: Phylogenetic studies and domain analysis of proteases were carried out to understand the molecular evolution as well as gene and protein features. Structural analysis was carried out to explore the binding mode and affinity of PIs for cognate proteases and prolyl oligopeptidase protease with inhibitor ligand. Results: In the study reported here, a significant number of proteases and PIs were identified in chickpea genome. The gene expression profiles of proteases and Pis in five different plant tissues revealed a differential expression pattern in more than one plant tissue. Molecular dynamics studies revealed the formation of stable complex owing to increased number of protein-ligand and inter and intramolecular protein-protein hydrogen bonds. Discussion: The genome-wide identification, characterization, evolutionary understanding, gene expression, and structural analysis of proteases and Pis provide a framework for future analysis when defining their roles in stress response and developing a more stress tolerant variety of chickpea. (C) 2014 Elsevier Ltd. All rights reserved.

10aDomain10aGene expression10aGenome analysis10aHomology modelling10aMolecular docking10amolecular dynamics simulation10aMotif10aOrthologs10aPhylogeny1 aSharma, Ranu1 aSuresh, C., G. uhttps://library.ncl.res.in/content/genome-wide-identification-and-structure-function-studies-proteases-and-protease-002266nas a2200241 4500008004100000022001400041245011000055210006900165260007300234300000800307490000700315520137800322653001601700653002001716653001801736653002501754653002501779100002901804700002601833700001901859700002001878856012601898 2015 eng d a0141-813000aPenicillin V acylase from pectobacterium atrosepticum exhibits high specific activity and unique kinetics0 aPenicillin V acylase from pectobacterium atrosepticum exhibits h aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcAUG a1-70 v793 a

Penicillin V acylases (PVAs, E.C.3.5.11) belong to the Ntn hydrolase super family of enzymes that catalyze the deacylation of the side chain from phenoxymethyl penicillin (penicillin V). Penicillin acylases find use in the pharmaceutical industry for the production of semi-synthetic antibiotics. PVAs employ the N-terminal cysteine residue as catalytic nucleophile and are structurally and evolutionarily related to bile salt hydrolases (BSHs). Here, we report the cloning and characterization of a PVA enzyme from the Gram-negative plant pathogen, Pectobacterium atrosepticum (PaPVA). The enzyme was cloned and expressed in Escherichia coli attaining a very high yield (250 mg/l) and a comparatively high specific activity (430 IU/mg). The enzyme showed marginally better pH and thermo-stability over PVAs characterized from Gram-positive bacteria. The enzyme also showed enhanced activity in presence of organic solvents and detergents. The enzyme kinetics turned out to be significantly different from that of previously reported PVAs, displaying positive cooperativity and substrate inhibition. The presence of bile salts had a modulating effect on PaPVA activity. Sequence analysis and characterization reveal the distinctive nature of these enzymes and underscore the need to study PVAs from Gram-negative bacteria. (C) 2015 Elsevier B.V. All rights reserved.

10aCooperative10aEnzyme kinetics10aGram-negative10aPenicillin V acylase10aSubstrate inhibition1 aAvinash, Vellore, Sunder1 aRamasamy, Sureshkumar1 aSuresh, C., G.1 aPundle, Archana uhttps://library.ncl.res.in/content/penicillin-v-acylase-pectobacterium-atrosepticum-exhibits-high-specific-activity-and-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-102331nas a2200241 4500008004100000022001400041245010100055210006900156260009900225300001200324490000700336520143200343653002601775653001501801653002401816653002001840653001701860653002501877100001801902700002601920700001901946856012401965 2016 eng d a1359-511300aHighly active bile salt hydrolase from enterococcus faecalis shows positive cooperative kinetics0 aHighly active bile salt hydrolase from enterococcus faecalis sho aTHE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, OXON, ENGLANDbELSEVIER SCI LTDcFEB a263-2690 v513 a

Bile salt hydrolase (BSH; cholylglycine hydrolase, EC 3.5.1.24) is an important enzyme that catalyses the deconjugation of bile acids conjugated with glycine or taurine and assists in the reduction of blood cholesterol levels. In the present study, we report the cloning, overexpression and characterisation of BSH gene from a gut-associated microbe Enterococcus faecalis (EfBSH). The overexpressed protein in Escherichia coli was purified to homogeneity. Optimum pH and temperature for activity were found to be 5.0 and 50 degrees C, respectively. The enzyme was considerably stable in the pH range of 5.0-7.0 and at a temperature of up to 50 degrees C. It showed high specific activity of 1390 U mg(-1) and 1289 U mg-1 for substrates such as glycocholic acid (GCA) and glycodeoxycholic acid (GDCA), respectively. The effect of additives on enzyme activity was assessed, and the detergent Triton X showed a marginally enhanced activity. The enzyme demonstrated unique enzyme kinetics of non-linear regression, thereby displaying positive cooperativity. In addition, the modulating effect of the non-substrate ligand Pen V on the hydrolysing ability of EfBSH towards bile acid such as GDCA was measured. It was observed that Pen V significantly enhanced the BSH activity. This is markedly different from the previously reported competitive inhibition of BSH activity by Pen V. (C) 2015 Elsevier Ltd. All rights reserved.

10aAllosteric regulation10aBile acids10aBile salt hydrolase10aEnzyme kinetics10aGut organism10aPenicillin V acylase1 aChand, Deepak1 aRamasamy, Sureshkumar1 aSuresh, C., G. uhttps://library.ncl.res.in/content/highly-active-bile-salt-hydrolase-enterococcus-faecalis-shows-positive-cooperative-002253nas a2200181 4500008004100000022001400041245014600055210006900201260009100270300002900361490000700390520147700397100001601874700001901890700001901909700001901928856012401947 2016 eng d a1932-620300aTwo chitotriose-specific lectins show anti-angiogenesis, induces caspase-9-mediated apoptosis and early arrest of pancreatic tumor cell cycle0 aTwo chitotriosespecific lectins show antiangiogenesis induces ca a1160 BATTERY STREET, STE 100, SAN FRANCISCO, CA 94111 USAbPUBLIC LIBRARY SCIENCEcJAN aArticle Number: e01461100 v113 a

The antiproliferative activity of two chito-specific agglutinins purified from Benincasa hispida (BhL) and Datura innoxia (DiL9) of different plant family origin was investigated on various cancer cell lines. Both lectins showed chitotriose specificity, by inhibiting lectin hemagglutinating activity. On further studies, it was revealed that these agglutinins caused remarkable concentration-dependent antiproliferative effect on human pancreatic cancerous cells but not on the normal human umbilical vein endothelial cells even at higher doses determined using MTT assay. The GI(50) values were approximately 8.4 mu g ml(-1) (0.247 mu M) and 142 mu g ml(-1) (14.8 mu M) for BhL and DiL9, respectively, against PANC-1 cells. The growth inhibitory effect of these lectins on pancreatic cancer cells were shown to be a consequence of lectin cell surface binding and triggering G(0)/G(1) arrest, mitochondrial membrane depolarization, sustained increase of the intracellular calcium release and the apoptotic signal is amplified by activation of caspases executing cell death. Interestingly, these lectins also showed anti-angiogenic activity by disrupting the endothelial tubulogenesis. Therefore, we report for the first time two chito-specific lectins specifically binding to tumor glycans; they can be considered to be a class of molecules with antitumor activity against pancreatic cancer cells mediated through caspase dependent mitochondrial apoptotic pathway.

1 aSingh, Ruby1 aNawale, Laxman1 aSarkar, Dhiman1 aSuresh, C., G. uhttps://library.ncl.res.in/content/two-chitotriose-specific-lectins-show-anti-angiogenesis-induces-caspase-9-mediated-102355nas a2200193 4500008004100000245007700041210006900118260000800187300001400195490000900209520173400218100001401952700002001966700001401986700001902000700001902019700001702038856010602055 2017 eng d00aMolecular features of bile salt hydrolases and relevance in human health0 aMolecular features of bile salt hydrolases and relevance in huma cJAN a2981-29910 v18613 aBackground: Bile salt hydrolase (BSH) enzyme is responsible for the de-conjugation of bile salts by commensal bacteria, thus playing a vital role in their colonization and survival in the mammalian intestine and determination of their probiotic potential. Further, bile deconjugation also leads to lowering of cholesterol and alterations in energy homeostasis, thus making BSH a clinically important enzyme. Scope of the review: Many recent observations have indicated that BSH may be involved in a multifaceted array of roles, directly or indirectly in the host and microbial physiology. BSH paralogues have now been found to occur in different microbes including free-living and pathogenic bacteria and Archaea. BSHs from various sources also show differential activity and substrate spectrum. Certain bacteria are known to possess multiple genes for BSH enzymes. BSHs have been reported to influence different metabolic phenomena, including bacterial pathogenesis and the maintenance of lipid and glucose homeostasis in the host. These observations necessitate an intense study into the biochemical, structural and regulatory features of BSH enzymes to better understand their role in regulating bacterial and host metabolism. Major conclusions: In this review, the available information on the characteristics of BSH enzymes have been organized in order to understand their interactions with a wide range of substrates and their myriad physiological roles, from bile resistance to signalling mechanisms. General significance: A detailed exploration of BSH architecture and regulation could provide insights into its evolution and a deeper appreciation of the multiple functions of this enzyme relevant to healthcare.1 aChand, D.1 aAvinash, V., S.1 aYadav, Y.1 aPundle, A., V.1 aSuresh, C., G.1 aRamasamy, S. uhttps://library.ncl.res.in/content/molecular-features-bile-salt-hydrolases-and-relevance-human-health02072nas a2200205 4500008004100000022001500041245010100056210006900157260000800226300001400234490000800248520136500256100002201621700001901643700001901662700001901681700002501700700002101725856012001746 2018 eng d a 0141-813000aActivation of HRI is mediated by Hsp90 during stress through modulation of the HRI-Hsp90 complex0 aActivation of HRI is mediated by Hsp90 during stress through mod cOCT a1604-16130 v1183 aHeme Regulated Inhibitor (HRI) is known to get activated in various stresses such as heme deficiency, heat shock, heavy metal toxicity etc. Heat shock protein 90 (Hsp90), a ubiquitous cytoplasmic protein interacts with HRI in order to regulate protein synthesis. However, it still remains to establish this interaction of HRI and Hsp90 at cellular levels and how this modulation of HRI activity is mediated by Hsp90 during stress. In the present report, using co-immunoprecipitation analysis we show that HRI interacts with Hsp90 and this association is independent of other co-chaperones in in vitro conditions. Further, analysis using truncated domains of HRI revealed that the K1 subdomain is essential for HRI - Hsp90 complex formation. Our in silico protein - protein interaction studies also indicated interaction of Hsp90 with K1 subdomain of HRI. Mammalian two hybrid assay validated this HRI - Hsp90 interaction at cellular levels. When the in vitro kinase assay was carried out with the co-immunoprecipitated complex of HRI - Hsp90, an increase in the kinase activity was observed resulting elevated levels of eIF2 alpha phosphorylation upon heavy metal stress and heat shock. Thus, our results clearly indicate modulation of HRI kinase activity with simultaneous Hsp90 association under stress conditions. (C) 2018 Elsevier B.V. All rights reserved.1 aBerwal, Sunil, K.1 aBhatia, Varsha1 aBendre, Arneya1 aSuresh, C., G.1 aChatterjee, Sangeeta1 aPal, Jayanta, K. uhttps://library.ncl.res.in/content/activation-hri-mediated-hsp90-during-stress-through-modulation-hri-hsp90-complex01872nas a2200157 4500008004100000245009900041210006900140260000800209300001200217490000800229520128800237100002201525700002601547700001901573856012201592 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. uhttps://library.ncl.res.in/content/analysis-kunitz-inhibitors-plants-comprehensive-structural-and-functional-insights03019nas a2200265 4500008004100000022001300041245014800054210006900202260000800271300001200279490000900291520209300300653002902393653002402422653002302446653001602469653002602485653002602511100001402537700001802551700001702569700001702586700001902603856013102622 2018 eng d a1570963900aStructure and function of a highly active Bile Salt Hydrolase (BSH) from Enterococcus faecalis and post-translational processing of BSH enzymes0 aStructure and function of a highly active Bile Salt Hydrolase BS cJAN a507-5180 v18663 aBile Salt Hydrolase (BSH), a member of Cholylglycine hydrolase family, catalyzes the de-conjugation of bile acids and is evolutionarily related to penicillin V acylase (PVA) that hydrolyses a different substrate such as penicillin V. We report the three-dimensional structure of a BSH enzyme from the Gram-positive bacteria Enterococcus faecalis (EfBSH) which has manifold higher hydrolase activity compared to other known BSHs and displays unique allosteric catalytic property. The structural analysis revealed reduced secondary structure content compared to other known BSH structures, particularly devoid of an anti-parallel β-sheet in the assembly loop and part of a β-strand is converted to increase the length of a substrate binding loop 2. The analysis of the substrate binding pocket showed reduced volume owing to altered loop conformations and increased hydrophobicity contributed by a higher ratio of hydrophobic to hydrophilic groups present. The aromatic residues F18, Y20 and F65 participate in substrate binding. Thus, their mutation affects enzyme activity. Docking and Molecular Dynamics simulation studies showed effective polar complementarity present for the three hydroxyl (-OH) groups of GCA substrate in the binding site contributing to higher substrate specificity and efficient catalysis. These are unique features characteristics of this BSH enzyme and thought to contribute to its higher activity and specificity towards bile salts as well as allosteric effects. Further, mechanism of autocatalytic processing of Cholylglycine Hydrolases by the excision of an N-terminal Pre-peptide was examined by inserting different N-terminal pre-peptides in EfBSH sequence. The results suggest that two serine residues next to nucleophile cysteine are essential for autocalytic processing to remove precursor peptide. Since pre-peptide is absent in EfBSH the mutation of these serines is tolerated. This suggests that an evolution-mediated subordination of the pre-peptide excision site resulted in loss of pre-peptide in EfBSH and other related Cholylglycine hydrolases.10aautocatalytic processing10aBile salt hydrolase10amolecular dynamics10aMutagenesis10aPolar complementarity10aX-ray crystallography1 aChand, D.1 aPanigrahi, P.1 aVarshney, N.1 aRamasamy, S.1 aSuresh, C., G. uhttps://library.ncl.res.in/content/structure-and-function-highly-active-bile-salt-hydrolase-bsh-enterococcus-faecalis-and-post01833nas a2200241 4500008004100000022001400041245012600055210006900181260000800250300001400258490000700272520099600279653001301275653002201288653002001310653002901330653001201359100002201371700001901393700002801412700002601440856012501466 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 uhttps://library.ncl.res.in/content/structural-insights-unique-inhibitory-mechanism-kunitz-type-trypsin-inhibitor-cicer-0