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Increased structural flexibility at the active site of a fluorophore-conjugated beta-lactamase distinctively impacts its binding toward diverse cephalosporin antibiotics.

ABSTRACT: The ?-loop at the active site of ?-lactamases exerts significant impact on the kinetics and substrate profile of these enzymes by forming part of the substrate binding site and posing as steric hindrance toward bulky substrates. Mutating certain residues on the ?-loop has been a general strategy for molecular evolution of ?-lactamases to expand their hydrolytic activity toward extended-spectrum antibiotics through a mechanism believed to involve enhanced structural flexibility of the ?-loop. Yet no structural information is available that demonstrates such flexibility or its relation to substrate profile and enzyme kinetics. Here we report an engineered ?-lactamase that contains an environment-sensitive fluorophore conjugated near its active site to probe the structural dynamics of the ?-loop and to detect the binding of diverse substrates. Our results show that this engineered ?-lactamase has improved binding kinetics and positive fluorescence signal toward oxyimino-cephalosporins, but shows little such effect to non-oxyimino-cephalosporins. Structural studies reveal that the ?-loop adopts a less stabilized structure, and readily undergoes conformational change to accommodate the binding of bulky oxyimino-cephalosporins while no such change is observed for non-oxyimino-cephalosporins. Mutational studies further confirm that this substrate-induced structural change is directly responsible for the positive fluorescence signal specific to oxyimino-cephalosporins. Our data provide mechanistic evidence to support the long-standing model that the evolutionary strategy of mutating the ?-loop leads to increased structural flexibility of this region, which in turn facilitates the binding of extended spectrum ?-lactam antibiotics. The oxyimino-cephalosporin-specific fluorescence profile of our engineered ?-lactamase also demonstrates the possibility of designing substrate-selective biosensing systems.

SUBMITTER: Wong WT

PROVIDER: S-EPMC3173089 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Increased structural flexibility at the active site of a fluorophore-conjugated beta-lactamase distinctively impacts its binding toward diverse cephalosporin antibiotics.

Wong Wai-Ting WT Chan Kwok-Chu KC So Pui-Kin PK Yap Hong-Kin HK Chung Wai-Hong WH Leung Yun-Chung YC Wong Kwok-Yin KY Zhao Yanxiang Y

The Journal of biological chemistry 20110623 36

The Ω-loop at the active site of β-lactamases exerts significant impact on the kinetics and substrate profile of these enzymes by forming part of the substrate binding site and posing as steric hindrance toward bulky substrates. Mutating certain residues on the Ω-loop has been a general strategy for molecular evolution of β-lactamases to expand their hydrolytic activity toward extended-spectrum antibiotics through a mechanism believed to involve enhanced structural flexibility of the Ω-loop. Yet ...[more]

PMID: 21705325

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Project description:Hypermutation due to DNA mismatch repair (MMR) deficiencies can accelerate the development of antibiotic resistance in Pseudomonas aeruginosa. Whether hypermutators generate resistance through predominantly similar molecular mechanisms to wild-type (WT) strains is not fully understood. Here, we show that MMR-deficient P. aeruginosa can evolve resistance to important broad-spectrum cephalosporin/beta-lactamase inhibitor combination antibiotics through novel mechanisms not commonly observed in WT lineages. Using whole-genome sequencing (WGS) and transcriptional profiling of isolates that underwent in vitro adaptation to ceftazidime/avibactam (CZA), we characterized the detailed sequence of mutational and transcriptional changes underlying the development of resistance. Surprisingly, MMR-deficient lineages rapidly developed high-level resistance (>256 μg/mL) largely without corresponding fixed mutations or transcriptional changes in well-established resistance genes. Further investigation revealed that these isolates had paradoxically generated an early inactivating mutation in the mexB gene of the MexAB-OprM efflux pump, a primary mediator of CZA resistance in P. aeruginosa, potentially driving an evolutionary search for alternative resistance mechanisms. In addition to alterations in a number of genes not known to be associated with resistance, 2 mutations were observed in the operon encoding the RND efflux pump MexVW. These mutations resulted in a 4- to 6-fold increase in resistance to ceftazidime, CZA, cefepime, and ceftolozane-tazobactam when engineered into a WT strain, demonstrating a potentially important and previously unappreciated mechanism of resistance to these antibiotics in P. aeruginosa. Our results suggest that MMR-deficient isolates may rapidly evolve novel resistance mechanisms, sometimes with complex dynamics that reflect gene inactivation that occurs with hypermutation. The apparent ease with which hypermutators may switch to alternative resistance mechanisms for which antibiotics have not been developed may carry important clinical implications.

Dataset Information

Increased structural flexibility at the active site of a fluorophore-conjugated beta-lactamase distinctively impacts its binding toward diverse cephalosporin antibiotics.

Publications

Increased structural flexibility at the active site of a fluorophore-conjugated beta-lactamase distinctively impacts its binding toward diverse cephalosporin antibiotics.

Similar Datasets

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