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

Abstract

e 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. Members of the carbapenem class of ␤-lactams are among the most important antimicrobial agents available for the treatment of serious bacterial infections (1). However, their use against Gram-negative bacteria is now threatened by the dissemination of carbapenemases, ␤-lactamases that hydrolyze the amide bond of the ␤-lactam ring, thereby inactivating carbapenems and other ␤-lactams (2). To date, only a few effective carbapenemase inhibitors have been available for clinical use. Enzymes with carbapenemase activity have been identified in multiple ␤-lactamase classes. ␤-Lactamases are divided into four classes, of which classes A, C, and D are serine enzymes that catalyze hydrolysis of the ␤-lactam through a serine-bound acyl intermediate, whereas class B ␤-lactamases, or metallo-␤-lactamases (MBLs), coordinate one or two zinc ions in the active site, which are essential for their enzymatic activity (1-3). The class B MBLs can be divided into four subclasses, according to their primary structure: B1a (e.g., VIM, IMP, DIM, and SPM), B1b (NDM), B2 (e.g., CphA), and B3 (e.g., L1 and AIM) (3). Currently, no clinically effective MBL inhibitor has been approved for use (4). Avibactam, the only clinically approved effective carbap...

Toxicity of β-Lactam Antibiotics: Pathophysiology, Molecular Biology and Possible Recovery Strategies

“…The class B MBLs can be divided into four subclasses according to their structure: (a) B1aVIM, imipenemase (IMP), DIM, SPM; (b) B1b: NDM, (c) B2: cphA, (d) B3: LI and AIM [37,60]. Moreover, Tripoli metallo-β-lactamase (TBM-1) was also included in MBLs [58,61].…”

“…For instance, GIM-1 MBL was reported in a clinical isolate (P. aeruginosa) in Germany in year 2002, irstly. In recent years, GIM-1 was started to be reported in other bacterial species such as S. marcescens, E. cloacae and Acinetobacter pitii [60,67]. Similarly, SIM-1 was obtained uncommonly and an integron-encoded blaSIM-1 was reported from Acinetobacter baumannii in Korea irstly [68].…”

Structural Insights into TMB-1 and the Role of Residues 119 and 228 in Substrate and Inhibitor Binding

“…Previous studies found that substitutions of residue 228 affected catalytic efficiency in, e.g., GIM-1 (25). Residue 228 has been thoroughly studied in several MBL enzymes; however, a proline variant similar to that found in TMB-2 has been described only in a VIM-2 R228P mutant (11).…”

“…Residue 228 has been thoroughly studied in several MBL enzymes; however, a proline variant similar to that found in TMB-2 has been described only in a VIM-2 R228P mutant (11). Residue 228 is located in MBL loop L3 (residues 223 to 240) and has been reported to contribute to substrate specificity (25,26) and to be involved in inhibitor binding (8,27).…”

“…Residue 228 is located adjacent to the active-site Zn2 in the so-called R2 binding site (36) and is a part of the MBL L3 loop. Substitutions at position 228 have been shown to be tolerated for different substrates (37) but have also been found to affect the substrate specificity of, e.g., GIM-1 (25). S228 in VIM-26 was reported to contribute to formation of a larger active-site pocket, leaving more space for substrates with larger R2 groups compared to, e.g., VIM-2 and VIM-7 with an arginine residue at this position (38).…”