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Role of Residues W228 and Y233 in the Structure and Activity of Metallo-?-Lactamase GIM-1.

ABSTRACT: Metallo-?-lactamases (MBLs) hydrolyze virtually all ?-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. The worldwide emergence of antibiotic-resistant bacteria harboring MBLs poses an increasing clinical threat. The MBL German imipenemase-1 (GIM-1) possesses an active site that is narrower and more hydrophobic than the active sites of other MBLs. The GIM-1 active-site groove is shaped by the presence of the aromatic side chains of tryptophan at residue 228 and tyrosine at residue 233, positions where other MBLs harbor hydrophilic residues. To investigate the importance of these two residues, eight site-directed mutants of GIM-1, W228R/A/Y/S and Y233N/A/I/S, were generated and characterized using enzyme kinetics, thermostability assays, and determination of the MICs of representative ?-lactams. The structures of selected mutants were obtained by X-ray crystallography, and their interactions with ?-lactam substrates were modeled in silico. Steady-state kinetics revealed that both positions are important to GIM-1 activity but that the effects of individual mutations vary depending on the ?-lactam substrate. Activity against type 1 substrates bearing electron-donating C-3/C-4 substituents (cefoxitin, meropenem) could be enhanced by mutations at position 228, whereas hydrolysis of type 2 substrates (benzylpenicillin, ampicillin, ceftazidime, imipenem) with methyl or positively charged substituents was favored by mutations at position 233. The crystal structures showed that mutations at position 228 or the Y233A variant alters the conformation of GIM-1 loop L1 rather than that of loop L3, on which the mutations are located. Taken together, these data show that point mutations at both positions 228 and 233 can influence the catalytic properties and the structure of GIM-1.

SUBMITTER: Skagseth S

PROVIDER: S-EPMC4750685 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1.

Skagseth Susann S Carlsen Trine Josefine TJ Bjerga Gro Elin Kjæreng GE Spencer James J Samuelsen Ørjan Ø Leiros Hanna-Kirsti S HK

Antimicrobial agents and chemotherapy 20151207 2

Metallo-β-lactamases (MBLs) hydrolyze virtually all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. The worldwide emergence of antibiotic-resistant bacteria harboring MBLs poses an increasing clinical threat. The MBL German imipenemase-1 (GIM-1) possesses an active site that is narrower and more hydrophobic than the active sites of other MBLs. The GIM-1 active-site groove is shaped by the presence of the aromatic side chains of tryptophan at residue 228 and tyrosine ...[more]

PMID: 26643332

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Project description:Metallo-β-lactamase (MBL)-producing Enterobacteriaceae are of grave clinical concern, particularly as there are no metallo-β-lactamase inhibitors approved for clinical use. The discovery and development of MBL inhibitors to restore the efficacy of available β-lactams are thus imperative. We investigated a zinc-chelating moiety, 1,4,7-triazacyclononane (TACN), for its inhibitory activity against clinical carbapenem-resistant Enterobacteriaceae MICs, minimum bactericidal concentrations (MBCs), the serum effect, fractional inhibitory concentration indexes, and time-kill kinetics were determined using broth microdilution techniques according to Clinical and Laboratory Standards Institute (CSLI) guidelines. Enzyme kinetic parameters and the cytotoxic effects of TACN were determined using spectrophotometric assays. The interactions of the enzyme-TACN complex were investigated by computational studies. Meropenem regained its activity against carbapenemase-producing Enterobacteriaceae, with the MIC decreasing from between 8 and 64 mg/liter to 0.03 mg/liter in the presence of TACN. The TACN-meropenem combination showed bactericidal effects with an MBC/MIC ratio of ≤4, and synergistic activity was observed. Human serum effects on the MICs were insignificant, and TACN was found to be noncytotoxic at concentrations above the MIC values. Computational studies predicted that TACN inhibits MBLs by targeting their catalytic active-site pockets. This was supported by its inhibition constant (Ki ), which was 0.044 μM, and its inactivation constant (Kinact), which was 0.0406 min-1, demonstrating that TACN inhibits MBLs efficiently and holds promise as a potential inhibitor.IMPORTANCE Carbapenem-resistant Enterobacteriaceae (CRE)-mediated infections remain a significant public health concern and have been reported to be critical in the World Health Organization's priority pathogens list for the research and development of new antibiotics. CRE produce enzymes, such as metallo-β-lactamases (MBLs), which inactivate β-lactam antibiotics. Combination therapies involving a β-lactam antibiotic and a β-lactamase inhibitor remain a major treatment option for infections caused by β-lactamase-producing organisms. Currently, no MBL inhibitor-β-lactam combination therapy is clinically available for MBL-positive bacterial infections. Hence, developing efficient molecules capable of inhibiting these enzymes could be a promising way to overcome this phenomenon. TACN played a significant role in the inhibitory activity of the tested molecules against CREs by potentiating the activity of carbapenem. This study demonstrates that TACN inhibits MBLs efficiently and holds promises as a potential MBL inhibitor to help curb the global health threat posed by MBL-producing CREs.

Dataset Information

Role of Residues W228 and Y233 in the Structure and Activity of Metallo-?-Lactamase GIM-1.

Publications

Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1.

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