Structural milestones in the reaction pathway of an amide hydrolase: substrate,acyl, and product complexes of cephalothin with AmpC beta-lactamase |
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Authors: | Beadle Beth M Trehan Indi Focia Pamela J Shoichet Brian K |
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Institution: | Department of Molecular Pharmacology and Biological Chemistry, Northwestern University, Chicago, Illinois 60611, USA. |
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Abstract: | Beta-lactamases hydrolyze beta-lactam antibiotics and are the leading cause of bacterial resistance to these drugs. Although beta-lactamases have been extensively studied, structures of the substrate-enzyme and product-enzyme complexes have proven elusive. Here, the structure of a mutant AmpC in complex with the beta-lactam cephalothin in its substrate and product forms was determined by X-ray crystallography to 1.53 A resolution. The acyl-enzyme intermediate between AmpC and cephalothin was determined to 2.06 A resolution. The ligand undergoes a dramatic conformational change as the reaction progresses, with the characteristic six-membered dihydrothiazine ring of cephalothin rotating by 109 degrees. These structures correspond to all three intermediates along the reaction path and provide insight into substrate recognition, catalysis, and product expulsion. |
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