Structural analysis of a penicillin V acylase from Pectobacterium atrosepticum confirms the importance of two Trp residues for activity and specificity

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• 作者：Vellore Sunder Avinash ; Priyabrata Panigrahi ; Deepak Chand ; Archana Pundle ; Cheravakattu Gopalan Suresh ; Sureshkumar Ramasamy ; ^{s.ramasamy@ncl.res.in" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Cholylglycine hydrolase ; Oligomeric interactions ; Molecular dynamics ; Mutagenesis ; Aromatic stacking
• 刊名：Journal of Structural Biology
• 出版年：2016
• 出版时间：February 2016
• 年：2016
• 卷：193
• 期：2
• 页码：85-94
• 全文大小：2492 K

文摘

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 Å 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.