01922nas a2200265   4500008004100000022001400041245011800055210006900173260009500242300001200337490000800349520092800357653002401285653001201309653002301321653001501344653001901359653002301378100002901401700002601430700001901456700002401475700002601499856013101525       2013                        eng d  a0006-291X00aStructural modelling of substrate binding and inhibition in penicillin V acylase from pectobacterium atrosepticum0 aStructural modelling of substrate binding and inhibition in peni  a525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USAbACADEMIC PRESS INC ELSEVIER SCIENCEcAUG  a538-5430 v4373 a<p>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.</p>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-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<p>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.</p>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-002266nas a2200277   4500008004100000022001400041245007800055210006900133260009200202300001200294490000700306520130400313653001601617653002001633653001701653653001901670653001801689653001901707653001501726653001501741100002901756700002801785700002601813700003501839856011401874       2016                        eng d  a0738-855100aPenicillin acylases revisited: importance beyond their industrial utility0 aPenicillin acylases revisited importance beyond their industrial  a4 PARK SQUARE, MILTON PARK, ABINGDON OX14 4RN, OXON, ENGLANDbTAYLOR & FRANCIS LTDcMAR  a303-3160 v363 a<p>It is of great importance to study the physiological roles of enzymes in nature; however, in some cases, it is not easily apparent. Penicillin acylases are pharmaceutically important enzymes that cleave the acyl side chains of penicillins, thus paving the way for production of newer semi-synthetic antibiotics. They are classified according to the type of penicillin (G or V) that they preferentially hydrolyze. Penicillin acylases are also used in the resolution of racemic mixtures and peptide synthesis. However, it is rather unfortunate that the focus on the use of penicillin acylases for industrial applications has stolen the spotlight from the study of the importance of these enzymes in natural metabolism. The penicillin acylases, so far characterized from different organisms, show differences in their structural nature and substrate spectrum. These enzymes are also closely related to the bacterial signalling phenomenon, quorum sensing, as detailed in this review. This review details studies on biochemical and structural characteristics of recently discovered penicillin acylases. We also attempt to organize the available insights into the possible in vivo role of penicillin acylases and related enzymes and emphasize the need to refocus research efforts in this direction.</p>10abeta-lactam10acholoyl glycine10ain vivo role10aNtn hydrolases10apathogenicity10aquorum sensing10aregulation10asignalling1 aAvinash, Vellore, Sunder1 aPundle, Archana, Vishnu1 aRamasamy, Sureshkumar1 aSuresh, Cheravakkattu, Gopalan  uhttps://library.ncl.res.in/content/penicillin-acylases-revisited-importance-beyond-their-industrial-utility-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<p>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.</p>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-002038nas a2200169   4500008004100000245015500041210006900196260000800265300001000273490000700283520134800290100002901638700002701667700002201694700002001716856013201736       2017                        eng d00aBiotransformation of penicillin V to 6-aminopenicillanic acid using immobilized whole cells of E. coli expressing a highly active penicillin V acylase0 aBiotransformation of penicillin V to 6aminopenicillanic acid usi  cJAN  a52-570 v473 aThe production of 6-aminopenicillanic acid (6-APA) is a key step in the manufacture of semisynthetic antibiotics in the pharmaceutical industry. The penicillin G acylase from Escherichia coli has long been utilized for this purpose. However, the use of penicillin V acylases (PVA) presents some advantages including better stability and higher conversion rates. The industrial application of PVAs has so far been limited due to the nonavailability of suitable bacterial strains and cost issues. In this study, whole-cell immobilization of a recombinant PVA enzyme from Pectobacterium atrosepticum expressed in E. coli was performed. Membrane permeabilization with detergent was used to enhance the cell-bound PVA activity, and the cells were encapsulated in calcium alginate beads and cross-linked with glutaraldehyde. Optimization of parameters for the biotransformation by immobilized cells showed that full conversion of pen V to 6-APA could be achieved within 1 hr at pH 5.0 and 35 degrees C, till 4% (w/v) concentration of the substrate. The beads could be stored for 28 days at 4 degrees C with minimal loss in activity and were reusable up to 10 cycles with 1-hr hardening in CaCl2 between each cycle. The high enzyme productivity of the PVA enzyme system makes a promising case for its application for 6-APA production in the industry.1 aAvinash, Vellore, Sunder1 aChauhan, Palna, Dinesh1 aGaikwad, Shraddha1 aPundle, Archana  uhttps://library.ncl.res.in/content/biotransformation-penicillin-v-6-aminopenicillanic-acid-using-immobilized-whole-cells-e-coli