Lefurgy S T, Malashkevich V N, Aguilan J T, Nieves E, Mundorff E C, Biju B, Noel M A, Toro R, Baiwir D, Papp-Wallace K M, Almo S C, Frere J-M, Bou G, Bonomo R A
Department of Chemistry, Hofstra University, Hempstead, New York, USA.
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA.
Antimicrob Agents Chemother. 2015 Nov 2;60(2):717-28. doi: 10.1128/AAC.01887-15. Print 2016 Feb.
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.
C类β-内酰胺酶对头孢霉素（如头孢西丁、头孢替坦和莫西沙星）的水解作用较弱。在过去20年中，一个新的基于质粒的赋予对头孢西丁耐药性的AmpCβ-内酰胺酶家族，即FOX家族，已发展到包括9个源自豚鼠气单胞菌染色体AmpC的独特成员。为了解FOX家族中独特的头孢霉素酶活性的基础，我们使用Y150F脱酰基缺陷变体，测定了FOX-4的第一个X射线晶体结构，即无配体酶及其与同名化合物头孢西丁形成的酰基酶结构。值得注意的是，N端标记的FOX-4的重组表达还产生了一种以前在β-内酰胺酶中未观察到的无活性腺苷酸化酶形式。这种发生在活性位点Ser64上的翻译后修饰（PTM）似乎没有提供选择优势，但可能为新型抗菌药物的设计提供了机会。在酰基酶复合物中可以看到配体诱导的酶的显著变化，特别是R2环和螺旋H10（P289至N297）区域，F293移动了10.3 Å。综上所述，本研究首次描绘了这种高效的C类头孢霉素酶，揭示了一种新的PTM，并提出了一种可能的头孢霉素耐药机制，该机制涉及由于配体结合口袋中存在S153P、N289P和N346I取代而导致底物重新定位。
