Refined crystal structure of β-lactamase from Citrobacter freundiiindicates a mechanism for β-lactam hydrolysis

BETA-LACTAMASES (EC 3.5.2.6, 'penicillinases') are a family of enzymes that protect bacteria against the lethal effects of cell-wall synthesis of penicillins, cephalosporins and related antibiotic agents, by hydrolysing the β-lactam antibiotics to biologically inactive compounds. Their production can, therefore, greatly contribute to the clinical problem of antibiotic resistance1–4. Three classes of β-lactamases—A, B and C—have been identified on the basis of their amino-acid sequence; class B β-lactamases are metalloenzymes, and are clearly distinct from members of class A and Cβ-lactamases5, which both contain an active-site serine residue involved in the formation of an acyl enzyme with β-lactam substrates during catalysis6–12. It has been predicted that class C β-lactamases share common structural features with D,D-carboxypeptidases and class A β-lactamases, and further, suggested that class A and class Cβ-lactamases have the same evolutionary origin as other β-lactam target enzymes13,14. We report here the refined three-dimensional structure of the class C β-lactamase from Citrobacter freundii12,15 at 2.0-Å resolution and confirm the predicted structural similarity. The refined structure of the acyl-enzyme formed with the monobactam inhibitor aztreonam at 2.5-Å resolution defines the enzyme's active site and, along with molecular modelling, indicates a mechanism for β-lactam hydrolysis. This leads to the hypothesis that Tyr 150 functions as a general base during catalysis.

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