[en] Class C β-lactamases poorly hydrolyze cephamycins (e.g., cefoxitin, cefotetan, and moxalactam). In the past 2 decades, a new family of plasmid-based AmpC β-lactamases conferring resistance to cefoxitin, the FOX family, has grown to include nine unique members descended from the Aeromonas caviae chromosomal AmpC. To understand the basis for the unique cephamycinase activity in the FOX family, we determined the first X-ray crystal structures of FOX-4, apo enzyme and the acyl-enzyme with its namesake compound, cefoxitin, using the Y150F deacylation-deficient variant. Notably, recombinant expression of N-terminally tagged FOX-4 also yielded an inactive adenylylated enzyme form not previously observed in β-lactamases. The posttranslational modification (PTM), which occurs on the active site Ser64, would not seem to provide a selective advantage, yet might present an opportunity for the design of novel antibacterial drugs. Substantial ligand-induced changes in the enzyme are seen in the acyl-enzyme complex, particularly the R2 loop and helix H10 (P289 to N297), with movement of F293 by 10.3 Å. Taken together, this study provides the first picture of this highly proficient class C cephamycinase, uncovers a novel PTM, and suggests a possible cephamycin resistance mechanism involving repositioning of the substrate due to the presence of S153P, N289P, and N346I substitutions in the ligand binding pocket.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Lefurgy, S.T.; Hofstra University > Department of Chemistry
Malashkevich, V.N.; Albert Einstein College of Medecine > Department of Biochemistry
Aguilan, J.T.; Albert Einstein College of Medecine > Department of Biochemistry
Nieves, E.; Albert Einstein College of Medecine > Department of Biochemistry
Mundorff, E.C.; Hofstra University > Department of Chemistry
Biju, B.; Hofstra University > Department of Chemistry
Noel, M.A.; Hofstra University > Department of Chemistry
Toro, R; Albert Einstein College of Medecine > Department of Biochemistry
Papp-Wallace, K.M.; Louis Stokes Cleveland Veterans Affairs Medical Center > Research Service
Almo, S.C.; Albert Einstein College of Medecine > Department of Biochemistry
Frère, Jean-Marie ; Université de Liège > Département des sciences de la vie > Centre d'ingénierie des protéines
Bou, G.; Complejo Hospitalario Universitario A. Coruña > Servicio de Microbiologia
Bonomo, R.A.; Louis Stokes Cleveland Veterans Affairs Medical Center - Case Western Reserve University > Departments of Medecine, Pharmacology, Molecular Biology and Microbiology and Biochemistry > Research Service
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