CMY-30 and CMY-42 are extended-spectrum (ES) derivatives of CMY-2. ES characteristics are due to substitutions of Gly (CMY- 30) and Ser (CMY-42) for Val211 in the -loop. To characterize the effects of 211 substitutions, we studied the interactions of CMY-2, -30, and -42 with boronic acid transition state inhibitors (BATSIs) resembling ceftazidime and cefotaxime, assessed thermal stability of the enzymes in their free forms and in complexes with BATSIs and oximino- -lactams, and simulated, using molecular dynamics (MD), the CMY-42 apoenzyme and the CMY-42 complexes with ceftazidime and the ceftazidime-like BATSI. Inhibition constants showed that affinities between CMY-30 and CMY-42 and the R1 groups of BATSIs were lower than those of CMY-2. ES variants also exhibited decreased thermal stability either as apoenzymes or in covalent complexes with oxi- mino compounds. MD simulations further supported destabilization of the ES variants. Val211Ser increased thermal factors of the -loop backbone atoms, as previously observed for CMY-30. The similar effects of the two substitutions seemed to be due to a less-constrained Tyr221 likely inducing concerted movement of elements at the edges of the active site ( -loop–Q120 loop–R2 loop/H10 helix). This inner-protein movement, along with the wider R1 binding cleft, enabled intense vibrations of the cova- lently bound ceftazidime and ceftazidime-like BATSIs. Increased flexibility of the ES enzymes may assist the productive adapta- tion of the active site to the various geometries of the oximino substrates during the reaction (higher frequency of near-attack conformations).

Interactions of Oxyimino-Substituted Boronic Acids and β-Lactams with the CMY-2-Derived Extended-Spectrum Cephalosporinases CMY-30 and CMY-42 / S., Kotsakis; Caselli, Emilia; L., Tzouvelekis; E., Petinaki; Prati, Fabio; V., Miriagou. - In: ANTIMICROBIAL AGENTS AND CHEMOTHERAPY. - ISSN 0066-4804. - STAMPA. - 57:(2013), pp. 968-976. [10.1128/AAC.01620-12]

Interactions of Oxyimino-Substituted Boronic Acids and β-Lactams with the CMY-2-Derived Extended-Spectrum Cephalosporinases CMY-30 and CMY-42

CASELLI, Emilia;PRATI, Fabio;
2013

Abstract

CMY-30 and CMY-42 are extended-spectrum (ES) derivatives of CMY-2. ES characteristics are due to substitutions of Gly (CMY- 30) and Ser (CMY-42) for Val211 in the -loop. To characterize the effects of 211 substitutions, we studied the interactions of CMY-2, -30, and -42 with boronic acid transition state inhibitors (BATSIs) resembling ceftazidime and cefotaxime, assessed thermal stability of the enzymes in their free forms and in complexes with BATSIs and oximino- -lactams, and simulated, using molecular dynamics (MD), the CMY-42 apoenzyme and the CMY-42 complexes with ceftazidime and the ceftazidime-like BATSI. Inhibition constants showed that affinities between CMY-30 and CMY-42 and the R1 groups of BATSIs were lower than those of CMY-2. ES variants also exhibited decreased thermal stability either as apoenzymes or in covalent complexes with oxi- mino compounds. MD simulations further supported destabilization of the ES variants. Val211Ser increased thermal factors of the -loop backbone atoms, as previously observed for CMY-30. The similar effects of the two substitutions seemed to be due to a less-constrained Tyr221 likely inducing concerted movement of elements at the edges of the active site ( -loop–Q120 loop–R2 loop/H10 helix). This inner-protein movement, along with the wider R1 binding cleft, enabled intense vibrations of the cova- lently bound ceftazidime and ceftazidime-like BATSIs. Increased flexibility of the ES enzymes may assist the productive adapta- tion of the active site to the various geometries of the oximino substrates during the reaction (higher frequency of near-attack conformations).
2013
57
968
976
Interactions of Oxyimino-Substituted Boronic Acids and β-Lactams with the CMY-2-Derived Extended-Spectrum Cephalosporinases CMY-30 and CMY-42 / S., Kotsakis; Caselli, Emilia; L., Tzouvelekis; E., Petinaki; Prati, Fabio; V., Miriagou. - In: ANTIMICROBIAL AGENTS AND CHEMOTHERAPY. - ISSN 0066-4804. - STAMPA. - 57:(2013), pp. 968-976. [10.1128/AAC.01620-12]
S., Kotsakis; Caselli, Emilia; L., Tzouvelekis; E., Petinaki; Prati, Fabio; V., Miriagou
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/937891
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