Structural milestones in the reaction pathway of an amide hydrolase: substrate, acyl, and product complexes of cephalothin with AmpC beta-lactamase.
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.
Beadle
Beth M
BM
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University, Chicago, Illinois 60611, USA.
Trehan
Indi
I
Focia
Pamela J
PJ
Shoichet
Brian K
BK
D000577:Amides / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins; D001665:Binding Sites; D002511:Cephalosporins / Q000737:chemistry / Q000378:metabolism; D002512:Cephalothin / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism*
D000577:Amides; D001426:Bacterial Proteins; D002511:Cephalosporins; D008024:Ligands; D006867:Hydrolases; C048795:AmpC beta-lactamases; D001618:beta-Lactamases; D002512:Cephalothin