02452nas 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-0