Chinese Chemical Letters  2014, Vol.25 Issue (10):1323-1326   PDF    
Activation free energy of Zn(Ⅱ), Co(Ⅱ) binding to metallo-β-lactamase ImiS
Xia Yang, Ya-Jun Zhou, Pei He, Yun-Hua Guo, Cong-Jun Liu, Ke-Wu Yang     
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi'an 710069, China
Abstract: In an effort to understand the recombination of a B2 metallo-β-lactamase (MβL), the binding of metals to apo-ImiS was studied by isothermal titration calorimetry and fluorescence spectra. The binding of Zn(Ⅱ), Co(Ⅱ) to apo-ImiS resulted in activation free energies ΔG6 values of 93.719 and 92.948 kJ mol-1, respectively, and increasing of fluorescence intensity at maxima emission of 340 nm.
Key words: Antibiotic resistant bacteria     Metallo-β-lactamases     Metalloprotein recombinant     Thermokinetic parameters    
1. 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 blactamases,which cleave the C-N 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 CoⅡ-ⅠmiS is 4-coordinate [12]. Steady-state kinetic studies revealed that CoⅡ-ⅠmiS had catalytic activity with Kcat of 255 S-1 and Km of 99 mmol/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 mmol/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 CoⅡ-ⅠmiS suggest that one histidine, one aspartic acid,and one cysteine residue coordinate to the metal ion in CoⅡ-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 ZnⅡ and CoⅡ binding to ImiS and fluorescence spectral changes caused by the binding.

Fig. 1. The proposed binding of ZnⅡ,CoⅡ to apo-ImiS [13].
2. Experimental

2.1. 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-26bImiS 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 dialyzing the enzyme sample versus 15 mmol/L HEPES,pH 6.5,containing 10 mmol/L EDTA at 4 ℃,followed by dialyzing versus the same buffer also containing 150 mmol/L NaCl to remove the EDTA. The sample was allowed to go through a Sephadex G-25 column to remove EDTA completely [17]. ZnⅡ had been removed from the enzyme. The ZnⅡ- and CoⅡ-substituted ImiS were prepared by adding 1 equiv. ZnSO4or CoCl2directly into the apo-ImiS,respectively,incubating on ice for over 1 h [18],dialyzing versus 2× 1 L of freshly 50 mmol/L HEPES, pH 6.5 at 4 ℃ over 6 h,and concentration,and they were used for spectroscopic studies. 2.2. Isothermal titration calorimetry The calorimetric titration was performed on a Micro-DSCIII (Setaram,France) microcalorimeter at 298.15 K. Before collection of the thermokinetic data,the enthalpy of KCl aqueous solution (spectral purity) was measured at 17.266 ± 0.074 kJ/mol at 298.15 K, which matches the literature value of 17.241 ± 0.018 kJ/mol [19]; the relative deviation is 0.14%. Standard a-Al2O3was used for calibration of the heat capacity,the sample mass was 320.6 mg,and the standard molar heat capacity Cp (a-Al2O3) was 79.44 kJ mol-1 K -1 at 298.15 K, which matches the literature value of 79.02 kJ mol-1 K -1 [20]; the relative deviation is 0.53%. These steps make sure that the calorimetric system is accurate and reliable.

After the above calibration,400 mL of 20 mmol/L apo-ImiS in 50 mmol/L Tris buffer,pH 7.0,was put into the bottom of the stainless steel sample cell. 160 mL of 50 mmol/L ZnSO4 or CoCl2 solution in the same buffer was injected into the cavity of the sample cell,which was separated from the bottom interspaces and sealed by a slidable rubber ring. Shortly after equilibrium,when the microcalorimeter reached the desired test conditions,the stainless steel shaft was pushed down. As a result,the protein was mixed with metal ions simultaneously,and the thermogram was recorded. 2.3. Fluorescence characterization Fluorescence spectra were collected on a HITACHI F-4500 spectrometer operating at room temperature. The samples were determined with an excitation wavelength at 280 nm and an emission wavelength at 340 nm. 3. 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,0.50-1.0 mmol/L native ImiS was dialyzed versus multiple changes of 15 mmol/L HEPES,pH 6.5,containing 10 mmol/L EDTA at 4 ℃. The EDTA was removed by dialysis. ICP measurements demonstrated the resulting enzyme contained less than 0.05 molar equivalents of ZnⅡ. The prepared apo-ImiS was identified by SDS-PAGE and is shown in Fig. 2. To prepare ZnⅡ and CoⅡ-substituted ImiS,1 equiv. ZnSO4or CoCl2solution in 50 mmol/L Tris buffer,pH 7.0 was added directly to a solution of apo-ImiS in same buffer,respectively.

Fig. 2. SDS-PAGE gel of metallo-b-lactamase ImiS and apo-ImiS purification. Lane 1, molecular weight markers; lane 2,boiled cell fraction of BL21(DE3) E. coli cells containing the pET26b-ImiS plasmid before induction; lane 3,boiled cell fraction of BL21(DE3) E. coli cells containing the pET26b-ImiS plasmid after a 3 h induction period with 1 mmol/L IPTG; lane 4,crude protein after ultrasonication and centrifugation; lane 5,crude protein after overnight dialysis; lane 6,the purified ImiS; and line 7,the purified apo-ImiS.

Fig. 3. MALDI-TOF spectrum of metallo-β-lactamase ImiS.

The binding of metal ions to apo-ImiS was characterized by isothermal titration calorimetry. A 400 mL,20 mmol/L apo-ImiS sample in 50 mmol/L Tris buffer,pH 7.0,was titrated with 160 mL of 50 mmol/L ZnSO4or CoCl2solution in the same buffer in the microcalorimeter at 298.15 K. The thermograms of protein folding/ recombination caused by ZnⅡ or CoⅡ binding to apo-ImiS were recorded and the collected experimental data are listed in Table 1. Based on the thermokinetic equations (1) and (2) [21],the calculated thermokinetic parameters are listed in Table 2.

where H0is the total heat of reaction (corresponding to the area under the T/K curve); Hi ,the reaction heat at some time t (corresponding to the area under the curve at time t); d Hi/dt,the rate of heat production at time t; k,rate constant; n,reaction order; R,gas constant; T,absolute temperature; N,Avogadro constant; h, Planck constant; ΔG0 ,activation free energy.
Table 1
Collected thermodynamic data of ZnⅡ- and CoⅡ-substituted ImiS at 298.15 K.

Table 2
The activation free energy of ZnⅡ,CoⅡ binding to apo-ImiS at 298.15 K.

The thermograms of 50 mmol/L apo-,ZnⅡ- and CoⅡ-substituted ImiS are shown in Fig. 4. The thermograms showed that the binding of ZnⅡ and CoⅡ to apo-ImiS is an exothermic reaction at 293.15 K. In investigation of recombinant of metalloproteins,the following thermodynamic parameters were obtained, the activation free energies ΔG0 of ZnⅡ and CoⅡ binding to ImiS is 93.719 and 92.948 kJ/mol,respectively,which are larger than those of imipenem hydrolysis with ImiS (86.4 kJ/mol),penicillin G hydrolysis with B1 subclasses MβL L1 (88.26 kJ/mol),and cefazolin with B1 subclasses MβL CcrA (88.03 kJ/mol) [21, 22], and the corresponding reaction rate constants k are 0.2363 × 10 -3 and 0.3225 × 10 -3 S -1 ,respectively.

Fig. 4. The thermograms of ZnⅡ- and CoⅡ-ⅠmiS. The thermograms were recorded at 298.15 K,20 mmol/L apo-ImiS samples in 50 mmol/L Tris buffer,pH 7.0 were titrated with ZnⅡ and CoⅡ ion,respectively.

The bindings of ZnⅡ and CoⅡ to ImiS were characterized by fluorescence spectra. The fluorescence spectra of 50 mmol/L apo-, ZnⅡ- and CoⅡ-substituted ImiS are shown in Fig. 5. It is clear that the apo-ImiS and metal-substituted ImiS have same maxima emission at 340 nm,while the fluorescence intensity of the metalloproteins is notably larger than that of the apo-protein.

Fig. 5. Fluorescence spectra of apo-,ZnⅡ- and CoⅡ-ⅠmiS. Fluorescence spectra were obtained using an excitation wavelength at 280 nm and emission wavelength at 340 nm. 15 mmol/L Tris,pH 7.0 was used as buffer blank.

Bacterial resistance has become a serious public health issue. Therefore,effectively evaluating β_-lactamases and probing the drug-resistant strains are extremely important. Accordingly,the fluorogenic and hydrogel based substrates have been developed as reporters for imaging the gene expression of β-lactamases in vitro and in vivo [23, 24, 25]. Our studies in this work reveal that the binding of ZnⅡ and CoⅡ to ImiS increases fluorescence intensity of the enzyme and changes the activation free energy,suggesting that the isothermal titration calorimetry could be used for evaluation of fluorescent sensing of transition metal ions to apo-proteins.

To date,the crystal structures of several MβLs,including CcrA and L1,have been solved [9],but the structure of ImiS has not yet been reported. In an effort to characterize the structure of ImiS, recent spectroscopic characterization studies of CoⅡ-ⅠmiS suggest that one histidine,one aspartic acid,and one cysteine residue coordinate to the metal ion in CoⅡ-ⅠmiS [13],while this study offered thermodynamic parameters of the binding. These thermodynamic data gained in this study will be valuable for the further mechanism investigation of MβLs. Previous studies showed that B2 subclasses MβLs CcrA and L1 tightly bind two ZnⅡ ions per monomer [26, 27]; we will further characterize the binding of metal ions to binuclear MβLs. 4. 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 ZnⅡ,CoⅡ to apo-ImiS resulted in an activation free energy ΔG0 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 evaluation of fluorescent sensing of transition metal ions to apo-proteins. 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).

[1] Z.G. Wang, W. Fast, A.M. Valentine, S.J. Benkovic, Metallo-b-lactamase: structure and mechanism, Curr. Opin. Chem. Biol. 3 (1999) 614-622.
[2] M.W. Crowder, J. Spencer, A.J. Vila, Metallo-b-lactamases: novel weaponry for antibiotic resistance in bacteria, Acc. Chem. Res. 39 (2006) 721-728.
[3] T.R. Walsh, M.A. Toleman, L. Poirel, P. Nordmann, Metallo-b-lactamases: the quiet before the storm? Clin. Microbiol. Rev. 18 (2005) 306-325.
[4] K. Bush, G.A. Jacoby, Updated functional classification of b-lactamases, Antimicrob. Agents Chemother. 54 (2010) 969-976.
[5] J.H. Toney, J.G. Moloughney, Metallo-beta-lactamase inhibitors: promise for the future? Curr. Opin. Invest. Drugs 5 (2004) 823-826.
[6] C. Bebrone, Metallo-β-lactamases (classification, activity, genetic organization, structure, zinc coordination) and their superfamily, Biochem. Pharmacol. 74 (2007) 1686-1701.
[7] K.M. Papp-Wallace, A. Endimiani, M.A. Taracila, R.A. Bonomo, Carbapenems: past, present, and future, Antimicrob. Agents Chemother. 55 (2011) 4943-4960.
[8] M.H. Valladares, A. Felici, G. Weber, et al., Zn(Ⅱ) dependence of the Aeromonas hydrophila AE036 metallo-β-lactamases activity and stability, Biochemistry 36 (1997) 11534-11541.
[9] N.O. Concha, B.A. Rasmussen, K. Bush, O. Herzberg, Crystal structure of the widespectrum binuclear zinc beta-lactamase from Bacteroides fragilis, Structure 4 (1996) 823-836.
[10] N. Sharma, Z.X. Hu, M.W. Crowder, B. Bennett, Conformational changes in the metallo-β-lactamases ImiS during the catalytic reaction: an EPR spectro-kinetic study of Co(Ⅱ)-spin label interactions, J. Am. Chem. Soc. 130 (2008) 8215-8222.
[11] A.L. Costello, N.P. Sharma, K.W. Yang, M.W. Crowder, D.L. Tierney, X-ray absorption spectroscopy of the zinc-binding sites in the class B2 metallo-β-lactamases ImiS from Aeromonas veronii bv. sobria, Biochemistry 45 (2006) 13650-13658.
[12] N.P. Sharma, C. Hajdin, S. Chandrasekar, et al., Mechanistic studies on the mononuclear Zn(Ⅱ)-containing metallo-β-lactamases ImiS from Aeromonas sobria, Biochemistry 45 (2006) 10729-10738.
[13] P.A. Crawford, K.W. Yang, N. Sharma, B. Bennett, M.W. Crowder, Spectroscopic studies on Co(Ⅱ)-substituted metallo-β-lactamases ImiS from Aeromonas veronii bv. sobria, Biochemistry 44 (2005) 5168-5176.
[14] L. Feng, K.W. Yang, L.S. Zhou, et al., N-heterocyclic dicarboxylic acids: broadspectrum inhibitors of metallo-β-lactamasess with Co-antibacterial effect against antibiotic-resistant bacteria, Bioorg. Med. Chem. Lett. 22 (2012) 5185-5189.
[15] P.A. Crawford, N. Sharma, S. Chandrasekar, et al., Over-expression, purification, and characterization of metallo-β-lactamases ImiS from Aeromonas veronii bv. Sobria, Protein Expr. Purif. 36 (2004) 272-279.
[16] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248-254.
[17] K.W. Yang, M.W. Crowder, A method for removing ethylenediaminetetraacetic acid from apo-ImiS, Anal. Biochem. 329 (2004) 342-344.
[18] Z.G. Wang, S.J. Benkovic, Purification, characterization, and kinetic studies of a soluble Bacteroides fragilis metallo-β-lactamases that provides multiple antibiotic resistance, J. Biol. Chem. 273 (1998) 22402-22408.
[19] V.K. Marthada, The enthalpy of solution of SRM 1655 (KCl) in H2O, J. Res. Nat. Bur. Stand. 85 (1980) 467-471.
[20] D.A. Ditmars, S. Ishihara, S.S. Chang, Enthalpy and heat-capacity standard reference material: synthetic sapphire (α-Al2O3) from 10 to 2250 K, J. Res. Nat. Bur. Stand. 87 (1982) 159-163.
[21] H.Z. Gao, Q. Yang, X.Y. Yan, et al., Exploring antibiotic resistant mechanism by microcalorimetry determination of thermokinetic parameters of metallo-β-lactamases L1 catalyzing penicillin G hydrolysis, J. Therm. Anal. Calorim. 107 (2012) 321-324.
[22] L. Zhai, K.W. Yang, C.C. Liu, et al., Exploring antibiotic resistant mechanism by microcalorimetry. III: Determination of thermokinetic parameters of cefazolin hydrolysis with metallo-β-lactamases CcrA, J. Therm. Anal. Calorim. 111 (2013) 1657-1661.
[23] W.Z. Gao, B.G. Xing, R.Y. Tsien, J.H. Rao, Novel fluorogenic substrates for imaging b-lactamase gene expression, J. Am. Chem. Soc. 125 (2003) 11146-11147.
[24] Z. Yang, P.L. Ho, G. Liang, et al., Using b-lactamase to trigger supramolecular hydrogelation, J. Am. Chem. Soc. 129 (2007) 266-267.
[25] S. Mizukami, S. Watanabe, Y. Hori, K. Kikuchi, Covalent protein labeling based on noncatalytic b-lactamase and a designed FRET substrate, J. Am. Chem. Soc. 131 (2009) 5016-5017.
[26] M.D. Peraro, A.J. Vila, P. Carloni, M.L. Klein, Role of zinc content on the catalytic efficiency of B1 metallo-β-lactamases, J. Am. Chem. Soc. 129 (2007) 2808-2816.
[27] J.H. Ullah, T.R. Walsh, I.A. Taylor, et al., The crystal structure of the L1 metallo-blactamase from Stenotrophomonas maltophilia at 1.7 Å resolution, J. Mol. Biol. 284 (1998) 125-136..