01732nas a2200229 4500008004100000022001400041245015100055210006900206260007900275300001000354490000700364520082100371653002501192653002201217653002601239653003201265100002101297700002201318700002001340700001901360856012301379 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. uhttp://library.ncl.res.in/content/structural-and-active-site-modification-studies-implicate-glu-trp-and-arg-activity-001979nas a2200217 4500008004100000022001400041245016600055210006900221260010300290300001600393490000800409520106400417100001801481700002501499700002101524700002401545700002001569700002401589700001901613856012901632 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. uhttp://library.ncl.res.in/content/structural-and-functional-analysis-conjugated-bile-salt-hydrolase-bifidobacterium-longum-002045nas a2200265 4500008004100000022001400041245009700055210006900152260007500221300001200296490000700308520112300315653001601438653001701454653001801471653001301489653002101502653002301523653002101546100001901567700001901586700002601605700002501631856012301656 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. uhttp://library.ncl.res.in/content/steady-state-and-time-resolved-fluorescence-studies-hemagglutinin-moringa-oleifera-001651nas a2200217 4500008004100000022001400041245008300055210006900138260007300207300001200280490000700292520085400299653002301153653003501176653002401211100001901235700001901254700002601273700002501299856010901324 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. uhttp://library.ncl.res.in/content/structure-activity-relationship-hemagglutinin-moringa-oleifera-seeds-001921nas a2200265 4500008004100000022001400041245011800055210006900173260009500242300001200337490000800349520092800357653002401285653001201309653002301321653001501344653001901359653002301378100002901401700002601430700001901456700002401475700002601499856013001525 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 uhttp://library.ncl.res.in/content/structural-modelling-substrate-binding-and-inhibition-penicillin-v-acylase-pectobacterium-000711nas a2200205 4500008004100000022001400041245008800055210006900143260000800212300000900220490000700229653002400236653001600260653002600276100001800302700002600320700002600346700001900372856011400391 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. uhttp://library.ncl.res.in/content/structure-highly-active-bsh-enzyme-subordinated-post-translational-excision02735nas a2200277 4500008004100000022001400041245013100055210006900186260008000255300001400335490000700349520172600356653001702082653002802099653002302127653002102150653001402171653002302185653002002208100002602228700001802254700002202272700002602294700001902320856011802339 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. uhttp://library.ncl.res.in/content/sequence-and-structure-based-comparative-analysis-assess-identify-and-improve-003097nas a2200289 4500008004100000022001400041245018200055210006900237260006700306300001400373490000700387520197200394653002802366653002902394653002702423653002402450653002402474653001802498100001802516700002702534700002602561700002602587700002502613700002702638700001902665856012302684 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. uhttp://library.ncl.res.in/content/structure-mediation-substrate-binding-and-post-translational-processing-penicillin-103018nas a2200265 4500008004100000022001300041245014800054210006900202260000800271300001200279490000900291520209300300653002902393653002402422653002302446653001602469653002602485653002602511100001402537700001802551700001702569700001702586700001902603856013002622 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. uhttp://library.ncl.res.in/content/structure-and-function-highly-active-bile-salt-hydrolase-bsh-enterococcus-faecalis-and-post01832nas a2200241 4500008004100000022001400041245012600055210006900181260000800250300001400258490000700272520099600279653001301275653002201288653002001310653002901330653001201359100002201371700001901393700002801412700002601440856012401466 2019 eng d a0739-110200aStructural insights into the unique inhibitory mechanism of Kunitz type trypsin inhibitor from Cicer arietinum L. Vol. 370 aStructural insights into the unique inhibitory mechanism of Kuni cJUL a2669-26770 v373 a

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

10aChickpea10acrystal structure10ainhibitory loop10aKunitz trypsin inhibitor10aTrypsin1 aBendre, Ameya, D.1 aSuresh, C., G.1 aShanmugam, Dhanasekaran1 aRamasamy, Sureshkumar uhttp://library.ncl.res.in/content/structural-insights-unique-inhibitory-mechanism-kunitz-type-trypsin-inhibitor-cicer-0