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.

FIG 1

Compounds used in this study and other cephamycins. Cefoxitin is shown in blue, the 7-α-methoxyl group is shown in green, and the carboxamide side chain that is eliminated upon acylation of S64 is shown in red.

FIG 2

Multiple-sequence alignment of mature-length class C β-lactamases. Alignment was carried out using Clustal Omega. Conserved active-site motifs are shaded blue. The R2 loop is shaded yellow. Sequence motifs at positions 153 and 346 are shaded by residue type in magenta or green.

FIG 3

MS/MS spectrum of the FOX-4 triply charged tryptic peptide 55 to 68 (VSEQTLFEIGSVSK) at m/z 618.39. Peptides containing phosphoadenosine (PhosphoAd) display the characteristic neutral loss of 347.1 Da. The ion at m/z 754.0 represents the doubly charged phosphoadenosine neutral loss from the precursor ion. The matching b- and y-ion series are labeled.

FIG 4

UV-difference spectrum of adenylylated and unmodified FOX-4. Absorption spectra were collected for concentration-matched (A₂₈₀) samples from the mAPBA column flowthrough (adenylyl-FOX-4) and elution (unmodified FOX-4) that had been dialyzed into 25 mM Tris (pH 7.5). Inset: difference spectrum showing absorption due to the presence of adenylyl-S64.

FIG 5

Model of adenylyl-FOX-4. Adenylate was manually positioned in FOX-4 using the FOX-4/SM23 boronic acid transition state analogue structure (unpublished data) to guide the placement of phosphate prior to energy minimization. Gray dashes indicate polar contacts, and yellow dashes indicate π-π interactions.

FIG 6

The reaction proceeds through the reversible formation of the Henri-Michaelis complex (ES), followed by acylation to form a covalent intermediate (ES*) and deacylation to yield product (P). In general, class C enzymes are deacylation rate limited, so the steady-state rate constant k_cat approximates k₃ (34).

FIG 7

Crystal structure of apo-FOX-4 (1.6 Å) shows a cluster of water molecules (red spheres) interacting with residues R148, E272, G286, and N287 (yellow). Conserved active-site motif side chains are shown for SXXK_64–67, YXN_150–152, and KTG_315–317 (magenta). This image and those that follow were made using PyMOL (Schrödinger).

FIG 8

Overlay of wild-type FOX-4 (green) and Y150F FOX-4 (pink) apo crystal structures.

FIG 9

Overlay of wild-type FOX-4 (green) and Y150F FOX-4–acyl-cefoxitin (orange) crystal structures.

FIG 10

Structural changes in loops upon cefoxitin binding. Wild-type FOX-4, green; Y150F FOX-4–cefoxitin, orange. (A) R2 loop. (B) Ω loop.

FIG 11

Overlays of cephalosporins and cephamycins in class C β-lactamases. (A) Moxalactam in E. coli AmpC (wild type, PDB ID 1FCO chain B, gray; N152A, PDB ID 1I5Q chain B, orange). (B) Moxalactam (PDB ID 1FCO, gray) and cephalothin (PDB ID 1KVM, green) in wild-type E. coli AmpC. (C) Acyl-cephalothin (PDB ID 1KVM, green) and cephalothin product (PDB ID 1KVL, yellow) in wild-type E. coli AmpC. (D) Cefoxitin in FOX-4 (Y150F, PDB ID 5CGX, this work, cyan) and E. coli AmpC (N152G, PDB ID 4KEN, magenta).