Studies on the Inactivation of Enterobacter cloacae P99 Class C beta-Lactamase by Carbapenems: Doripenem, Panipenem, Imipenem and Biapenem　Structure-Function of Beta-Lactamases　Monday, Sep 21, 2015, 8:30 AM -11:00 AM

Interscience Conference of Antimicrobial Agents and Chemotherapy 2015 (ICAAC/ICC2015),San Diego, CA　San Diego Convention Center

Background: Class C beta-lactamases are widely disseminated in many Gram-negative bacteria and are among the most important determinants of beta-lactam resistance. Carbapenems have been effective against pathogens bearing class C beta-lactamases as antibiotics and beta-lactamase inhibitors. Methods: P99 beta-lactamase was expressed in Escherichia coli AS226-51 and purified by m-amino phenylborate affinity column, HiTrapSP cation exchange column and Superdex 200 Gel-filtration column. Kinetics, mass spectrometry, and crystallography were performed. Results: P99 beta-lactamase was crystallized in space group P21212 with one molecule in the asymmetric unit by vapor diffusion method. Atomic structures of P99 beta-lactamases complex with doripenem (dori) and panipenem (pani), imipenem (imi) and biapenem (bia) (were solved to 1.02, 1.15, 1.37 and 1.15 Å resolution, respectively. Mass spectrometry was performed in each case. Using mass spectrometry, the four carbapenems acylated P99. By x-ray crystallography, the carbonyl oxygen of three carbapenems, dori, pani and imi, were placed in oxyanion hole, differing from AmpC-imi complex (PDB:1ll5). The bia complex was formed in the oxyanion hole in the analogous fashion of AmpC-imi complex. The C2 carbon in the five-membered ring of dori is seen in the sp3 configuration and pani, imi and bia in the sp2, suggesting there is delta 1 pyrroline ring for dori acyl-enzyme and delta 2 for pani, imi and bia (Delta 1-Delta 2 tautomerization). Conclusions: In contrast to the AmpC beta-lactamase:imi complex (oxyanion hole is broken), both the P99 beta-lactamase:dori, :pani and : imi complexes show the carbonyl in the oxyanion hole. On the other hand, bia forms a broken oxyanion hole. All the acyl intermediates are very stable. These results suggest that two kinds of intermediates can be formed in the path of carbapenem reaction scheme with class C beta-lactamases.
M. Nukaga, N. Ohuchi, C. R. Bethel, R. A. Bonomo
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