Hydroxyl groups in the betabeta sandwich of metallo-beta-lactamases favor enzyme activity: Tyr218 and Ser262 pull down the lid.
Metallo-beta-lactamases (MBLs) efficiently hydrolyze and thereby inactivate various beta-lactam antibiotics in clinical use. Their potential to evolve into more efficient enzymes threatens public health. Recently, we have identified the designed F218Y mutant of IMP-1 as an enzyme with superior catalytic efficiency compared to the wild-type. Thus, it may be found in clinical isolates in the future. In an effort to elucidate the molecular mechanisms involved in enhanced activity, we carried out molecular dynamics simulations of ten MBL variants in complex with a cefotaxime intermediate. The stability of these near-transition state enzyme-substrate intermediate complexes was modeled and compared to the experimental catalytic efficiencies k(cat)/K(M). For each of the ten complexes ten independent simulations were performed. In each simulation the temperature was gradually increased and determined upon breakdown of the complex. Rankings based on the experimental catalytic efficiencies and the data from computer simulations were in good agreement. From trajectory analysis of stable simulations, the combination of Tyr218 and Ser262 was found to lead to an altered hydrogen bonding network, which translates into a closing down movement of a beta-hairpin loop covering the active site. These observations may explain the significantly decreased K(M) and increased k(cat)/K(M) values of this variant toward all substrates recently tested in experiment. Previously, we have discovered that mutations G262S (yielding IMP-1) and G262A in IMP-6 stabilize the Zn(II) ligand His263 and thus the enzyme-substrate intermediate complex through a domino effect, which enhances conversion of drugs like ceftazidime, penicillins, and imipenem. Together, the domino effect and the altered beta-hairpin loop conformation explain how IMP-6 can evolve through mutations G262S and F218Y into an enzyme with up to one order of magnitude increased catalytic efficiencies toward these important antibiotics. Furthermore, the previously proposed binding of a third zinc ion close to the active site of IMP-6 mutant S121G was corroborated by our simulations.
Oelschlaeger
Peter
P
Department of Chemistry, SGM 418, University of Southern California, 3620 McClintock Avenue, Los Angeles, CA 90089-1062, USA. poelschl@usc.edu
Pleiss
Juergen
J
D002384:Catalysis; D003198:Computer Simulation; D004789:Enzyme Activation; D006860:Hydrogen Bonding; D006878:Hydroxides / Q000737:chemistry*; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry; D015032:Zinc / Q000737:chemistry*; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics
D006878:Hydroxides; D008670:Metals; D014443:Tyrosine; D012694:Serine; C031356:hydroxide ion; D001618:beta-Lactamases; D015032:Zinc