Dataset Information

Structural Characterization of the D179N and D179Y Variants of KPC-2 β-Lactamase: Ω-Loop Destabilization as a Mechanism of Resistance to Ceftazidime-Avibactam.

ABSTRACT: Klebsiella pneumoniae carbapenemases (KPC-2 and KPC-3) present a global clinical threat, as these β-lactamases confer resistance to carbapenems and oxyimino-cephalosporins. Recent clinically identified KPC variants with substitutions at Ambler position D179, located in the Ω loop, are resistant to the β-lactam/β-lactamase inhibitor combination ceftazidime-avibactam, but susceptible to meropenem-vaborbactam. To gain insights into ceftazidime-avibactam resistance conferred by D179N/Y variants of KPC-2, crystal structures of these variants were determined. The D179N KPC-2 structure revealed that the change of the carboxyl to an amide moiety at position 179 disrupted the salt bridge with R164 present in wild-type KPC-2. Additional interactions were disrupted in the Ω loop, causing a decrease in the melting temperature. Shifts originating from N179 were also transmitted toward the active site, including ∼1-Å shifts of the deacylation water and interacting residue N170. The structure of the D179Y KPC-2 β-lactamase revealed more drastic changes, as this variant exhibited disorder of the Ω loop, with other flanking regions also being disordered. We postulate that the KPC-2 variants can accommodate ceftazidime because the Ω loop is displaced in D179Y or can be more readily displaced in D179N KPC-2. To understand why the β-lactamase inhibitor vaborbactam is less affected by the D179 variants than avibactam, we determined the crystal structure of D179N KPC-2 in complex with vaborbactam, which revealed wild-type KPC-2-like vaborbactam-active site interactions. Overall, the structural results regarding KPC-2 D179 variants revealed various degrees of destabilization of the Ω loop that contribute to ceftazidime-avibactam resistance, possible substrate-assisted catalysis of ceftazidime, and meropenem and meropenem-vaborbactam susceptibility.

SUBMITTER: Alsenani TA

PROVIDER: S-EPMC9017313 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Structural Characterization of the D179N and D179Y Variants of KPC-2 β-Lactamase: Ω-Loop Destabilization as a Mechanism of Resistance to Ceftazidime-Avibactam.

Alsenani T A TA Viviani S L SL Kumar V V Taracila M A MA Bethel C R CR Barnes M D MD Papp-Wallace K M KM Shields R K RK Nguyen M H MH Clancy C J CJ Bonomo R A RA van den Akker F F

Antimicrobial agents and chemotherapy 20220328 4

Klebsiella pneumoniae carbapenemases (KPC-2 and KPC-3) present a global clinical threat, as these β-lactamases confer resistance to carbapenems and oxyimino-cephalosporins. Recent clinically identified KPC variants with substitutions at Ambler position D179, located in the Ω loop, are resistant to the β-lactam/β-lactamase inhibitor combination ceftazidime-avibactam, but susceptible to meropenem-vaborbactam. To gain insights into ceftazidime-avibactam resistance conferred by D179N/Y variants of K ...[more]

PMID: 35341315

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Project description:KPC-2 is the most prevalent class A carbapenemase in the world. Previously, KPC-2 was shown to hydrolyze the β-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam. In addition, substitutions at amino acid position R220 in the KPC-2 β-lactamase increased resistance to clavulanic acid. A novel bridged diazabicyclooctane (DBO) non-β-lactam β-lactamase inhibitor, avibactam, was shown to inactivate the KPC-2 β-lactamase. To better understand the mechanistic basis for inhibition of KPC-2 by avibactam, we tested the potency of ampicillin-avibactam and ceftazidime-avibactam against engineered variants of the KPC-2 β-lactamase that possessed single amino acid substitutions at important sites (i.e., Ambler positions 69, 130, 234, 220, and 276) that were previously shown to confer inhibitor resistance in TEM and SHV β-lactamases. To this end, we performed susceptibility testing, biochemical assays, and molecular modeling. Escherichia coli DH10B carrying KPC-2 β-lactamase variants with the substitutions S130G, K234R, and R220M demonstrated elevated MICs for only the ampicillin-avibactam combinations (e.g., 512, 64, and 32 mg/liter, respectively, versus the MICs for wild-type KPC-2 at 2 to 8 mg/liter). Steady-state kinetics revealed that the S130G variant of KPC-2 resisted inactivation by avibactam; the k2/K ratio was significantly lowered 4 logs for the S130G variant from the ratio for the wild-type enzyme (21,580 M(-1) s(-1) to 1.2 M(-1) s(-1)). Molecular modeling and molecular dynamics simulations suggested that the mobility of K73 and its ability to activate S70 (i.e., function as a general base) may be impaired in the S130G variant of KPC-2, thereby explaining the slowed acylation. Moreover, we also advance the idea that the protonation of the sulfate nitrogen of avibactam may be slowed in the S130G variant, as S130 is the likely proton donor and another residue, possibly K234, must compensate. Our findings show that residues S130 as well as K234 and R220 contribute significantly to the mechanism of avibactam inactivation of KPC-2. Fortunately, the emergence of S130G, K234R, and R220M variants of KPC in the clinic should not result in failure of ceftazidime-avibactam, as the ceftazidime partner is potent against E. coli DH10B strains possessing all of these variants.

Dataset Information

Structural Characterization of the D179N and D179Y Variants of KPC-2 β-Lactamase: Ω-Loop Destabilization as a Mechanism of Resistance to Ceftazidime-Avibactam.

Publications

Structural Characterization of the D179N and D179Y Variants of KPC-2 β-Lactamase: Ω-Loop Destabilization as a Mechanism of Resistance to Ceftazidime-Avibactam.

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