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Structural insights into the loss of penicillinase and the gain of ceftazidimase activities by OXA-145 β-lactamase inPseudomonas aeruginosa

Abstract : Objectives We previously described extended-spectrum oxacillinase OXA-145 from Pseudomonas aeruginosa, which differs from narrow-spectrum OXA-35 by loss of Leu-155. The deletion results in loss of benzylpenicillin hydrolysis and acquisition of activity against ceftazidime. We report the crystal structure of OXA-145 and provide the basis of its switch in substrate specificity. Methods OXA-145 variants were generated by site-directed mutagenesis and purified to homogeneity. The crystal structure of OXA-145 was determined and molecular dynamics simulations were performed. Kinetic parameters were investigated in the absence and in the presence of sodium hydrogen carbonate (NaHCO3) for representative substrates. Results The structure of OXA-145 was obtained at a resolution of 2.3 Å and its superposition with that of OXA-10 showed that Trp-154 was shifted by 1.8 Å away from the catalytic Lys-70, which was not N-carboxylated. Addition of NaHCO3 significantly increased the catalytic efficiency against penicillins, but not against ceftazidime. The active-site cavity of OXA-145 was larger than that of OXA-10, which may favour the accommodation of large molecules such as ceftazidime. Molecular dynamics simulations of OXA-145 in complex with ceftazidime revealed two highly coordinated water molecules on the α- or β-face of the acyl ester bond, between Ser-67 and ceftazidime, which could be involved in the catalytic process. Conclusions Deletion of Leu-155 resulted in inefficient positioning of Trp-154, leading to a non-carboxylated Lys-70 and thus to loss of hydrolysis of the penicillins. Ceftazidime hydrolysis could be attributed to enlargement of the active site and to a catalytic mechanism independent of the carboxylated Lys-70.
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Submitted on : Thursday, January 13, 2022 - 5:45:30 PM
Last modification on : Thursday, April 7, 2022 - 10:10:50 AM

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D. Meziane-Cherif, R. Bonnet, A. Haouz, P. Courvalin. Structural insights into the loss of penicillinase and the gain of ceftazidimase activities by OXA-145 β-lactamase inPseudomonas aeruginosa. Journal of Antimicrobial Chemotherapy, Oxford University Press (OUP), 2015, 71 (2), pp.395 - 402. ⟨10.1093/jac/dkv375⟩. ⟨pasteur-03525333⟩

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