The Drug-Resistant Variant P167S Expands the Substrate Profile of CTX-M β-Lactamases for Oxyimino-Cephalosporin Antibiotics by Enlarging the Active Site upon Acylation

Journal Article · Wed Jun 14 00:00:00 EDT 2017 · Biochemistry

DOI:https://doi.org/10.1021/acs.biochem.7b00176· OSTI ID:1418300

Patel, Meha P. ^[1]; Hu, Liya ^[2]; Stojanoski, Vlatko ^[3]; Sankaran, Banumathi ^[4]; Prasad, B. V. Venkataram ^[2]; Palzkill, Timothy  ^[5]

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1. Baylor College of Medicine, Houston, TX (United States). Interdepartmental Graduate Program in Translational Biology and Molecular Medicine; Baylor College of Medicine, Houston, TX (United States). Dept. of Pharmacology
2. Baylor College of Medicine, Houston, TX (United States). Verna Marrs McLean Dept. of Biochemistry and Molecular Biology
3. Baylor College of Medicine, Houston, TX (United States). Dept. of Pharmacology; Baylor College of Medicine, Houston, TX (United States). Verna Marrs McLean Dept. of Biochemistry and Molecular Biology
4. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Berkeley Center for Structural Biology, Dept. of Molecular Biophysics and Integrated Bioimaging
5. Baylor College of Medicine, Houston, TX (United States). Interdepartmental Graduate Program in Translational Biology and Molecular Medicine; Baylor College of Medicine, Houston, TX (United States). Dept. of Pharmacology; Baylor College of Medicine, Houston, TX (United States). Verna Marrs McLean Dept. of Biochemistry and Molecular Biology

β-Lactamases are enzymes produced by bacterial cells that provide resistance to β-lactam antibiotics. The CTX-M class of β-lactamases provides resistance against the antibiotic, cefotaxime, but not a related oxyimino-cephalosporin antibiotic, ceftazidime. β-lactamases that carry the P167S substitution, however, have been reported to provide ceftazidime resistance. The mechanism by which the P167S substitution expands the substrate profile of CTX-M enzymes is not known. In this study, CTX-M-14 was used as the model enzyme to study the structural changes caused by the P167S mutation that may accelerate ceftazidime turnover. X-ray crystallography was used to determine the structures of the CTX-M-14 P167S apo-enzyme along with the structures of the S70G/P167S, E166A/P167S and E166A mutant enzymes complexed with ceftazidime as well as the E166A/P167S apo-enzyme. The S70G and E166A mutations allow the capture of the enzyme-substrate complex and acylated forms of the ceftazidime molecule, respectively. The results showed a large conformational change in the Ω-loop of the CTX-M-14 ceftazidime acyl-enzyme complex of the P167S mutant but not in the enzyme-substrate complex suggesting the conformational change occurs upon acylation. The conformational change results in a larger active site cavity that prevents steric clash between the aminothiazole ring of ceftazidime and the Asn170 residue in the Ω-loop, allowing for accommodation of ceftazidime for hydrolysis. In addition, the conformational change in the Ω-loop was not observed in the E166A/P167S apoenzyme, suggesting the presence of acylated ceftazidime influences the conformational change. Finally, the E166A acyl-enzyme structure with ceftazidime did not exhibit the altered Ω-loop conformation, indicating the P167S substitution is required for the change. Taken together, the results reveal that the P167S substitution and the presence of acylated ceftazidime both drive the structure towards a conformational change of the Ω-loop and that in CTX-M P167S enzymes found in drug-resistant bacteria this will lead to increased ceftazidime hydrolysis. Lastly, this study demonstrates how a naturally occurring substitution can dramatically alter the active site to expand the substrate profile of an enzyme due to antibiotic selective pressure.

• Research Organization: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institutes of Health (NIH)
• Grant/Contract Number: AC02-05CH11231; R01AI32956; Q1279
• OSTI ID: 1418300
• Journal Information: Biochemistry, Vol. 56, Issue 27; ISSN 0006-2960
• Publisher: American Chemical Society (ACS)
• Country of Publication: United States
• Language: English

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