title: Analysis of the Structure and Function of FOX-4 Cephamycinase.
abstract: 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.
authors.affiliation: Department of Chemistry, Hofstra University, Hempstead, New York, USA.
authors.forename: S T
authors.initials: ST
authors.lastname: Lefurgy
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: V N
authors.initials: VN
authors.lastname: Malashkevich
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: J T
authors.initials: JT
authors.lastname: Aguilan
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: E
authors.initials: E
authors.lastname: Nieves
authors.affiliation: Department of Chemistry, Hofstra University, Hempstead, New York, USA.
authors.forename: E C
authors.initials: EC
authors.lastname: Mundorff
authors.affiliation: Department of Chemistry, Hofstra University, Hempstead, New York, USA.
authors.forename: B
authors.initials: B
authors.lastname: Biju
authors.affiliation: Department of Chemistry, Hofstra University, Hempstead, New York, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Noel
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: R
authors.initials: R
authors.lastname: Toro
authors.affiliation: GIGA Proteomic Facility, Mass Spectrometry Laboratory GIGA-R, Université de Liège, Sart-Tilman, Liège, Belgium.
authors.forename: D
authors.initials: D
authors.lastname: Baiwir
authors.affiliation: Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio, USA Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Papp-Wallace
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: S C
authors.initials: SC
authors.lastname: Almo
authors.affiliation: Centre d'Ingénierie des Protéines, Institut de Chimie, Université de Liège, Sart-Tilman, Liège, Belgium.
authors.forename: J-M
authors.initials: JM
authors.lastname: Frere
authors.affiliation: Servicio de Microbiologìa, Complejo Hospitalario Universitario A. Coruña, La Coruña, Spain.
authors.forename: G
authors.initials: G
authors.lastname: Bou
authors.affiliation: Research Service, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio, USA Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio, USA Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, USA robert.bonomo@va.gov.
authors.forename: R A
authors.initials: RA
authors.lastname: Bonomo
mesh_terms: D057585:Aeromonas caviae / Q000187:drug effects; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D002440:Cefoxitin / Q000378:metabolism / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D020033:Protein Isoforms; D002440:Cefoxitin; C413413:fox-4 protein, E coli; C048795:AmpC beta-lactamases; D001618:beta-Lactamases