00712nas 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-excision02736nas 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-002452nas a2200265 4500008004100000022001400041245015600055210006900211260009500280300001000375490000800385520139300393653002201786653002801808653002301836653001601859653002801875100002901903700002601932700001801958700002001976700003401996700002602030856013002056 2016 eng d a1047-847700aStructural analysis of a penicillin V acylase from pectobacterium atrosepticum confirms the importance of two Trp residues for activity and specificity0 aStructural analysis of a penicillin V acylase from pectobacteriu a525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USAbACADEMIC PRESS INC ELSEVIER SCIENCEcFEB a85-940 v1933 a

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

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