Original articleActivation free energy of Zn(II), Co(II) binding to metallo-β-lactamase ImiS
Graphical abstract
Binding of Zn(II), Co(II) to apo-ImiS results in an activation free energy value of 92.948 and 93.908 kJ mol−1, respectively, and increasing of fluorescence intensity at maxima emission of 340 nm.
Introduction
β-Lactam antibiotics remain the most useful chemotherapeutic agents in the fight against bacterial infections. However, the overuse of these antibiotics in clinical settings has resulted in a large number of pathogenic bacteria which are resistant to almost all commonly-used antibiotics. The most common way for bacteria to become resistant to β-lactam antibiotics is to produce the β-lactamases, which cleave the CN bond of β-lactam ring and render the drugs ineffective [1], [2], [3], [4].
There have been more than 1000 distinct β-lactamases identified, and these enzymes have been categorized into classes A, B, C and D, based on their amino acid sequence homologies [4]. Class A, C, and D enzymes are collectively called serine-β-lactamases, and these enzymes use a common catalytic mechanism in which an active site serine nucleophilically attacks the β-lactam carbonyl, ultimately leading to a cleaved β-lactam ring. Class B enzymes, also called metallo-β-lactamases (MβLs), utilize either 1 or 2 equiv. of Zn(II) to catalyze the β-lactam hydrolysis [5]. MβLs have been further subgrouped into subclasses B1–B3, based on their amino acid sequence homologies and Zn(II) content [4], [5], [6].
MβL ImiS, a representative of the B2 subclass enzymes, preferentially hydrolyzes carbapenems, which have been called one of the “last resort” antibiotics [7], and exhibits maximal activity when bound to only one Zn(II) [8]. Given the enormous biomedical importance of ImiS, there have been a large number of studies of this enzyme [9], [10], [11], [12], [13], [14]. Spectroscopic studies indicated that the catalytic activity of ImiS required Zn(II) or Co(II) (in substituted ImiS) bound by a cysteine and a histidine residues, and the metal ion can adopt 4- or 5-fold coordination [10]. Our studies showed that the reaction of ImiS with imipenem leads to a product-bound species coordinated to Zn via a carboxylate group [11], EPR studies revealed that the metal ion in Co(II)-ImiS is 4-coordinate [12]. Steady-state kinetic studies revealed that Co(II)-ImiS had catalytic activity with Kcat of 255 S−1 and Km of 99 μmol/L when using imipenem as substrate [13], and inhibition studies showed that N-heterocyclic dicarboxylic acid is a competitive inhibitor of ImiS with Ki value of 7.1 μmol/L [14]. So far, the crystal structures of several MβLs including CcrA and L1 have been solved [9], but the structure of ImiS has not yet been reported. Although a large number of structural, mechanism, kinetic, and inhibition studies on ImiS have been conducted, there is no report on the thermodynamic parameters of metal ions binding to ImiS to date. Recent NMR studies of Co(II)-ImiS suggest that one histidine, one aspartic acid, and one cysteine residue coordinate to the metal ion in Co(II)-ImiS [13]. In order to investigate thermodynamic effects of the binding of metal ions to ImiS (Fig. 1), in this paper, we first determined the activation free energy of Zn(II) and Co(II) binding to ImiS and fluorescence spectral changes caused by the binding.
Section snippets
Preparation of apo- and metal-substituted ImiS
MβL ImiS was prepared as previously reported [15]. Briefly, a single colony of BL21(DE3) Escherichia coli containing pET-26b-ImiS was allowed to grow in LB medium containing kanamycin. Protein production was induced by adding of 1 mmol/L IPTG for 3 h; protein was purified by SP-Sepharose column eluted with a linear gradient of 0–500 mmol/L NaCl. ImiS concentrations were determined using Beer's law and an extinction coefficient of 37,250 L mol−1 cm−1 at 280 nm [16].
The apo-ImiS was prepared by
Results and discussion
MβL ImiS was over-expressed in BL21(DE3) E. coli cells by the procedure as previously described [15]. ImiS was purified with a SP-Sepharose column eluted with a linear gradient of 0–500 mmol/L NaCl in 50 mmol/L Tris, pH 7.0. The protein was identified by SDS-PAGE as shown in Fig. 2. The MALDI-TOF mass spectrum of isolated ImiS with the [M+H+]+ peak at 25,209.91 m/z is shown in Fig. 3, which matches what Crowder reported [15]. The yield of ImiS was 5 mg per liter culture. To prepare apo-ImiS,
Conclusion
Based on the overexpression and purification of ImiS, confirmation by MS, and preparation of apo-ImiS, we first characterized the binding of metals to apo-ImiS by isothermal titration calorimetry. The binding of Zn(II), Co(II) to apo-ImiS resulted in an activation free energy value of 93.719 and 92.948 kJ/mol, respectively. With the binding, the fluorescence intensity of ImiS increased at maxima emission of 340 nm, suggesting that the isothermal titration calorimetry has potential for
Acknowledgments
The authors gratefully thank Professor Michael Crowder at Miami University for plasmid pET-26b-ImiS. This work was supported by the National Natural Science Foundation of China (Nos. 21272186 and 81361138018).
References (27)
- et al.
Metallo-β-lactamase: structure and mechanism
Curr. Opin. Chem. Biol.
(1999) Metallo-β-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily
Biochem. Pharmacol.
(2007)- et al.
Crystal structure of the wide-spectrum binuclear zinc beta-lactamase from Bacteroides fragilis
Structure
(1996) - et al.
N-heterocyclic dicarboxylic acids: broad-spectrum inhibitors of metallo-β-lactamases with Co-antibacterial effect against antibiotic-resistant bacteria
Bioorg. Med. Chem. Lett.
(2012) - et al.
Over-expression, purification, and characterization of metallo-β-lactamase ImiS from Aeromonas veronii bv. Sobria
Protein Expr. Purif.
(2004) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)- et al.
A method for removing ethylenediaminetetraacetic acid from apo-ImiS
Anal. Biochem.
(2004) - et al.
Purification, characterization, and kinetic studies of a soluble Bacteroides fragilis metallo-β-lactamase that provides multiple antibiotic resistance
J. Biol. Chem.
(1998) - et al.
The crystal structure of the L1 metallo-b-lactamase from Stenotrophomonas maltophilia at 1.7 Å resolution
J. Mol. Biol.
(1998) - et al.
Metallo-β-lactamases: novel weaponry for antibiotic resistance in bacteria
Acc. Chem. Res.
(2006)
Metallo-β-lactamases: the quiet before the storm?
Clin. Microbiol. Rev.
Updated functional classification of β-lactamases
Antimicrob. Agents Chemother.
Metallo-beta-lactamase inhibitors: promise for the future?
Curr. Opin. Invest. Drugs
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Co-first author and they contributed equally to this work.