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Crystallographic studies of enzymes that bind penicillins: Penicillin G acylase and DD-peptidase

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  • Biophysics
  • General
  • Biology
  • Engineering


Most bacteria have a cell wall sacculus that provides the cell with structural integrity, shape, and protection from osmotic lysis. The sacculus is composed of a polymer called peptidoglycan, sometimes referred to as murein. Cross-link formation of the inter-peptide bridge in peptidoglycan is the last step in cell wall biosynthesis and is catalyzed by a variety of enzymes called DD-peptidases. DD-peptidases are the targets of β-lactam antibiotics such as penicillins and cephalosporins. β-Lactams are structurally similar to the natural substrate of DD-peptidase, x-D-Ala-D-Ala. Unlike the peptidoglycan substrates, β-lactams form long-lived acyl-enzyme intermediates with DD-peptidases that inhibit the enzymes and eventually leads to bacterial cell death. This important class of antibiotics represents the most widely used clinical antibiotics in the world today. Unfortunately bacterial resistance to β-lactam antibiotics is on the rise. Resistance is mostly due to the introduction of β-lactam destroying enzymes known as β-lactamases. The evolution of DD-peptidases from cell wall synthesizing enzymes to β-lactamases is evident through their structural and functional similarities. ^ Presented here are the crystallographic structures of a DD-peptidase from Streptomyces sp. R61 complexed with a cephalosporin that mimics peptidoglycan binding and a highly specific DD-peptide in both substrate and product forms. These complexes provide valuable mechanistic information of the enzymes reaction pathway. The acyl-enzyme complex is observed using the cephalosporin peptidoglycan mimic. The Henri-Michaelis complex is observed using inactivated enzyme and DD-peptide. Carboxypeptidation products are observed, still bound in the active site, using active enzyme, DD-peptide and excess D-ala. The structures show well-defined substrate binding grooves and a specificity subsite that may be present in other PBPs. The subsite is complementary to a portion of the natural cell wall substrate that varies among bacterial species. ^ Penicillin G acylase is an enzyme used in the commercial manufacture of β-lactam antibiotic precursors. The crystallographic structural studies of a mutant penicillin G acylase from Providencia rettgeri strain BRO1 are presented here. An explanation of the altered substrate specificity is proposed to be a Metα140Leu mutation. This information is useful for engineering enzymes with altered specificity that will be used in the manufacture of β-lactam antibiotics. ^

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