Analysis of the Structure and Function of FOX-4 Cephamycinase

Abstract

j Class C ␤-lactamases poorly hydrolyze cephamycins (e.g., cefoxitin, cefotetan, and moxalactam). In the past 2 decades, a new family of plasmid-based AmpC ␤-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 Nterminally tagged FOX-4 also yielded an inactive adenylylated enzyme form not previously observed in ␤-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.

Atomic‐Resolution Structure of a Class C β‐Lactamase and Its Complex with Avibactam

“…The overall structure of TRU-1 closely resembles that of previously reported class Ce nzymes,s uch as plasmid-encoded MOX-1 (RMSD upon Ca matching 0.78 ), [20] CMY-10 (RMSD 0.87 ), [21] FOX-4 (RMSD 0.69 ), [22] and the chromosomal P. aeruginosa AmpC (RMSD 0.95 ) [19] and E. cloacae P99 AmpC (RMSD 1.14 ). [23] The structure of TRU-1 shows as ulfate anion bound almost at full occupancy (70 %) within the active site ( Figure 3).…”

“…On the other hand, TRU‐1 has a phenylalanine at position 293 that is also present in FOX‐4, but replaced by a leucine in several class C enzymes, such as CMY‐10 and MOX‐1 and P. aeruginosa AmpC (Figure A). Structural studies performed on FOX‐4 highlighted the importance of this residue in driving the movement of the R2 loop upon substrate binding . Indeed, the structure of FOX‐4 deacylation‐deficient variant Y150F in complex with cefoxitin shows that Cα of Phe293 moves 2.5 Å toward the substrate with rotation of the side chain by 130°, which places it within 3.5 Å of the cefoxitin dihydrothiazine moiety (Figure B).…”

“… A) Active‐site view of the superimposition of TRU‐1 (gray cartoon) with other class C enzymes (only the R2 loop is shown for clarity in these enzymes). The different amino acids occupying positions 292 and 293 are displayed as sticks in all sequences and colored accordingly: TRU‐1 gray carbon atoms (Ser292, Phe293); FOX‐4 (PDB ID: https://www.rcsb.org/structure/5CGS) cyan carbon atoms (Ser292, Phe293); MOX‐1 (PDB ID: https://www.rcsb.org/structure/3W8K) green carbon atoms (Ile292, Leu293); CMY‐10 (PDB ID: https://www.rcsb.org/structure/1ZKJ) gold carbon atoms (Ile292, Leu293); P. aeruginosa AmpC (PDB ID: https://www.rcsb.org/structure/4GZB) pink carbon atoms (Ala292, Leu293). B) Active‐site view of the superimposition of TRU‐1 (gray cartoon, gray carbon atoms) with FOX‐4 (PDB ID: https://www.rcsb.org/structure/5CGS; R2 loop in cyan cartoon, cyan carbon atoms) and FOX‐4 deacylation‐deficient variant Y150F in complex with cefoxitin (PDB ID: https://www.rcsb.org/structure/5CGX; R2 loop in lilac cartoon, lilac carbon atoms).…”

“…The different amino acids occupying positions 292 and 293 are displayed as sticks in all sequences and colored accordingly: TRU‐1 gray carbon atoms (Ser292, Phe293); FOX‐4 (PDB ID: https://www.rcsb.org/structure/5CGS) cyan carbon atoms (Ser292, Phe293); MOX‐1 (PDB ID: https://www.rcsb.org/structure/3W8K) green carbon atoms (Ile292, Leu293); CMY‐10 (PDB ID: https://www.rcsb.org/structure/1ZKJ) gold carbon atoms (Ile292, Leu293); P. aeruginosa AmpC (PDB ID: https://www.rcsb.org/structure/4GZB) pink carbon atoms (Ala292, Leu293). B) Active‐site view of the superimposition of TRU‐1 (gray cartoon, gray carbon atoms) with FOX‐4 (PDB ID: https://www.rcsb.org/structure/5CGS; R2 loop in cyan cartoon, cyan carbon atoms) and FOX‐4 deacylation‐deficient variant Y150F in complex with cefoxitin (PDB ID: https://www.rcsb.org/structure/5CGX; R2 loop in lilac cartoon, lilac carbon atoms). C) Active‐site view of the superimposition of TRU‐1 (gray cartoon, gray carbon atoms) with its complex with avibactam (R2 loop in orange, orange carbon atoms), FOX‐4 (PDB ID: https://www.rcsb.org/structure/5CGS, R2 loop in cyan cartoon, cyan carbon atoms) and FOX‐4 deacylation‐deficient variant Y150F in complex with cefoxitin (PDB ID: https://www.rcsb.org/structure/5CGX, R2 loop in lilac cartoon, lilac carbon atoms).…”

“…B) Active‐site view of the superimposition of TRU‐1 (gray cartoon, gray carbon atoms) with FOX‐4 (PDB ID: https://www.rcsb.org/structure/5CGS; R2 loop in cyan cartoon, cyan carbon atoms) and FOX‐4 deacylation‐deficient variant Y150F in complex with cefoxitin (PDB ID: https://www.rcsb.org/structure/5CGX; R2 loop in lilac cartoon, lilac carbon atoms). C) Active‐site view of the superimposition of TRU‐1 (gray cartoon, gray carbon atoms) with its complex with avibactam (R2 loop in orange, orange carbon atoms), FOX‐4 (PDB ID: https://www.rcsb.org/structure/5CGS, R2 loop in cyan cartoon, cyan carbon atoms) and FOX‐4 deacylation‐deficient variant Y150F in complex with cefoxitin (PDB ID: https://www.rcsb.org/structure/5CGX, R2 loop in lilac cartoon, lilac carbon atoms).…”