The Structural Role of N170 in Substrate-assisted Deacylation in KPC-2 β-Lactamase

The Klebsiella pneumoniae carbapenemase 2 (KPC-2) is a widely disseminated β lactamase that readily confers resistance to antibiotics such as carbapenems, which are critical for the treatment of infections caused by multidrug and extensively drug (MDR, XDR) resistant bacteria. Disturbingly, amino acid substitutions in KPC-2 that have arisen in the clinic are observed to lead to the development of resistance to ceftazidime-avibactam, a preferred treatment for KPC bearing Gram-negative bacteria. The omega loop [R164-D179, (Ambler numbering System)] is a structural feature that is important for substrate binding and catalysis. E166, the general base, and N170 in the omega loop position a strategically located water molecule that assists in deacylation. Specific substitutions in this region (164-179) result in changes in the structure and function of the enzyme, leading to alterations in substrate specificity, decreased stability, and more recently observed, increased resistance to ceftazidime/avibactam. Using accelerated rare-event sampling well-tempered metadynamics simulations, we explored in detail the structural role of R164 and D179 variants that are described to confer ceftazidime/avibactam resistance. Using wt-metaD, the buried conformation of D179 substitutions produce a pronounced structural disorder in the omega loop, more than R164 mutants, where the crystallographic omega loop structure remains mostly intact. Our findings also reveal that the conformation of N170 plays an under appreciated role impacting drug binding and restricting deacylation. Metadynamics simulations further support the hypothesis that KPC-2 D179 variants employ substrate-assisted catalysis for ceftazidime hydrolysis, involving the ring amine of the aminothiazole group to promote deacylation and catalytic turnover. Moreover, the shift in the WT conformation of N170 contributes to reduced deacylation and altered spectrum of enzymatic activity.