Structure-based Design of Selective metallo-β-lactamase

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Metallo-β-lactamases catalyzes the hydrolysis of most β-lactam antibiotics including carbapenems, and there are currently no potent inhibitors of such enzymes. Our ultimate goal is to develop structure-based inhibitors of metallo-β-lactamases and we set two subthemes :(1)Preparation of apoenzyme of IMP-1 metallo-β-lactamase from Seratia marcescens.The apo-IMP-1 enzyme can be obtained by application of rather high temperature(30℃),high concentration of EDTA(50 mM)and the medium of pH 6.5(MOPS-NaOH, 50 mM, pH 6.5, 1.0 M NaCl containing 30% glycerol).Excess EDTA was removed by use of short gel filtration column (PD-10)at 4℃. The Zn(II)content of the prepared apo-IMP-1 enzyme was checked by atomic absorption spectrometry to be 0.076 per an IMP-1 molecule, indicating that the activity of apo-IMP-1 enzyme is less than 95 % of the untreated EDTA. In apo-IMP-1 prepared by this method, the enzymatic activity was perfectly recovered by the addition of a small excess of Zn(NO_3)_2・6H_2O.(2) Crystallization and crystallographic of VIM-2 metallo-β-lactamase from Pseudomonas aeruginosa Complexed with a Mercaptocarboxylate InhibitorRecently, we found rac-2-Phenylpropyl-3-mercaptopropionic acid, PhenylC3SH, was found to be a potent inhibitor of VIM-2. The structure of the VIM-2-PhenylC3SH complex was determined by X-ray crystallography to 2.3 A. The structure revealed that the thiol group of PhenylC3SH bridged to the two zinc (II) ions and the phenyl group interacted with Tyr67 (47) on loop1 near the active site, by π-π stacking interactions. The methylene group interacted with Phe61 (42) located at the bottom of loop1 though CH-π interactions. Dynamic movements were observed in Arg228 (185) and Asn233 (190) on loop2, compared with the native structure (PDB code: 1KO3). These results suggest that the above-mentioned four residues play important roles in the binding and recognition of inhibitors or substrates and in stabilizing a loop in the VIM-2 enzyme.