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Antimicrobial Agents and Chemotherapy, December 2007, p. 4512-4514, Vol. 51, No. 12
0066-4804/07/$08.00+0     doi:10.1128/AAC.00491-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Biochemical Characterization of SFC-1, a Class A Carbapenem-Hydrolyzing ß-Lactamase{triangledown}

Fátima Fonseca,1 Ana Cristina Sarmento,1 Isabel Henriques,1 Bart Samyn,2 Jozef van Beeumen,2 Pedro Domingues,3 Maria Rosário Domingues,3 Maria José Saavedra,4 and António Correia1*

Centre for Environmental and Marine Studies and Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal,1 Department of Biochemistry, Physiology and Microbiology, Laboratory of Protein Biochemistry and Protein Engineering, Ghent University, Ghent, Belgium,2 Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal,3 Department of Veterinary Science, Center of Studies in Animal and Veterinary Science, University of Trás-os-Montes e Alto Douro, 5001-801 Vila Real, Portugal4

Received 11 April 2007/ Returned for modification 22 June 2007/ Accepted 20 July 2007


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ABSTRACT
 
The carbapenem-hydrolyzing ß-lactamase SFC-1 from Serratia fonticola UTAD54 was overexpressed in Escherichia coli, purified, and characterized. The enzyme exhibited an apparent molecular mass of 30.5 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SFC-1 hydrolyzes penicillins, cephalosporins, aztreonam, and carbapenems and is inhibited by clavulanic acid, sulbactam, and tazobactam.


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TEXT
 
Most of the strains included in the species Serratia fonticola express both a chromosomally encoded extended-spectrum class A ß-lactamase and a species-specific AmpC ß-lactamase (3, 8). Previous studies have shown that S. fonticola UTAD54 additionally expresses a metalloenzyme (Sfh-I) (5) and a class A carbapenem-hydrolyzing ß-lactamase (SFC-1) (1), both chromosomally encoded. In this study, the gene encoding SFC-1 from S. fonticola UTAD54 was cloned and the purified protein was characterized.

The blaSFC-1 gene was amplified by PCR using primers SFCexp2_for (5'-GGATCCACATGTCACGCACCGGTCGACTG) and SFCexp2_rev (5'-GTGCTCGAGTTAGAAGCCGATAGACTTTCC). The amplified fragment, digested with BspLu11I and XhoI (Roche Diagnostics, Meylan, France), was cloned into the NcoI site of pET-26, and Escherichia coli BL21(DE3) cells (Novagen Inc., Madison, WI) were transformed with the plasmid.

Recombinant SFC-1 was purified from an uninduced culture of E. coli BL21(DE3)(pMF13) grown in Luria-Bertani medium supplemented with kanamycin at 25°C. The cells were harvested by centrifugation and resuspended in 10 mM sodium phosphate buffer. Clear supernatant was loaded onto a preequilibrated S-Sepharose column (Amersham Pharmacia Biotech) with 10 mM sodium phosphate buffer (pH 6.0). The proteins were eluted with a linear gradient of NaCl in the same buffer. Fractions presenting ß-lactamase activity, measured as the initial rate of hydrolysis of 100 µM ampicillin ({Delta}{varepsilon}232 [molar absorption coefficient at 232 nm] = –1,070 M–1 cm–1), were loaded onto a preequilibrated Superdex 75 column (Amersham Pharmacia Biotech) with 10 mM sodium phosphate buffer, pH 6.0. Elution was performed with the same buffer, and active fractions were collected. The protein content of each sample was determined with the Pierce bicinchoninic acid protein assay. Approximately 25 mg of purified enzyme was obtained per liter of culture. Protein was estimated to be >95% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the overall yield of the purification protocol was 82%. Isoelectric focusing of crude ß-lactamase extracts of S. fonticola UTAD54 and E. coli BL21(DE3)(pMF13) and of purified SFC-1 was performed with commercially prepared polyacrylamide gel plates (pH 3 to 9; Amersham Pharmacia Biotech) on a Phast system apparatus (Amersham Pharmacia Biotech). Focused ß-lactamases were detected by incubation with nitrocefin (Oxoid, Basingstoke, United Kingdom). The pI of SFC-1 was determined by comparison to those of ß-lactamases TEM-2 and SHV-5. SDS-PAGE analysis showed that purified SFC-1 migrates as a single band, but isoelectric focusing revealed two forms of purified SFC-1, with pIs of 7.6 and 8.2. Electrospray ionization-mass spectrometry with a Q-TOF 2 mass spectrometer (Micromass, Manchester, United Kingdom) confirmed the presence of two molecular forms of SFC-1, corresponding to 30,773 and 31,083 Da. The value of 30,773 Da is consistent with the predicted molecular mass of the mature protein (1). Truncated forms of ß-lactamases have been encountered in other bacteria, but those forms share similar hydrolytic properties and inhibitor sensitivities (10).

Purified enzyme was subjected to electrophoresis on a 15% SDS-PAGE gel and then electroblotted onto a polyvinylidene difluoride membrane. After staining, SFC-1 bands were excised and subjected to N- and C-terminal sequencing as described previously (6). N- and C-terminal sequences were ASQPPQV and IGF-COOH, respectively, showing that a 26-amino-acid leader sequence is removed to generate the mature SFC-1.

Initial hydrolysis rates were measured at 25°C in 50 mM sodium phosphate (pH 7.0) on an ULTROSPEC 2000 spectrophotometer (Amersham Pharmacia Biotech). Kinetic parameters (Table 1) were determined by recording the initial rates at different substrate concentrations and by fitting the experimental data to the Michaelis-Menten equation. Inhibition was measured after a 10-min preincubation of the enzyme with an inhibitor in 50 mM sodium phosphate buffer (pH 7.0) by using cephalothin (100 µM) as the substrate. Fifty percent inhibitory concentrations (IC50s) were determined from inhibition graphs of percent control activity versus the concentration of the inhibitor. The activities against penicillins, narrow-spectrum cephalosporins, and aztreonam were very high, while the activities against expanded-spectrum cephalosporins were much lower, a common feature of other class A carbapenem-hydrolyzing ß-lactamases (2, 4, 11, 12). The highest turnover rate was obtained for cephalothin (kcat, 280.4 s–1), followed by aztreonam (162 s–1) and ampicillin (155 s–1). Although SFC-1 showed strong hydrolytic activity against aztreonam, revealed by the kcat, the enzyme was shown to have low affinity for this substrate (Km, 484.3 µM), and therefore, a low catalytic efficiency compared to those obtained using penicillins, cephalothin, and imipenem as substrates was observed. The highest catalytic efficiencies were observed with cephalothin and penicillins, while expanded-spectrum cephalosporins appeared to be poor substrates. SFC-1 had the highest apparent affinities for meropenem and piperacillin (Km, 25.7 and 37.5 µM, respectively). Among carbapenems, SFC-1 hydrolyzed imipenem at rates eight times higher than those for meropenem (kcat, 54.2 and 6.5 s–1, respectively).


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  		TABLE 1. Kinetic parameters of purified SFC-1 ß-lactamase and comparison to other class A carbapenem-hydrolyzing ß-lactamasesa

An analysis of SFC-1 kinetic parameters showed that this enzyme has a broad substrate profile, similar to that of other carbapenamases of group 2f (2, 4, 11, 12). However, SFC-1 exhibits catalytic efficiencies and turnover rates for ceftazidime and cefoxitin somewhat higher than those demonstrated by IMI-1 and KPC-1 (Table 1). The turnover rate for ceftazidime (kcat, 2.1 s–1) was approximately 300 and 20 times higher than those of IMI-1 and KPC-1 (0.0068 and 0.1 s–1, respectively), resulting in much higher catalytic efficiencies (1,730 and 40 times higher, respectively). SFC-1 hydrolyzed cefoxitin more efficiently than IMI-1 and KPC-1 (8 and 27 times, respectively).

The activity of SFC-1 was inhibited by the class A enzyme inhibitors clavulanic acid (IC50, 72.8 µM), sulbactam (IC50, 22.7 µM), and tazobactam (IC50, 6.9 µM). In contrast to previously published data for IMI-1 and KPC-1, which are well inhibited by tazobactam (IC50, 0.03 and 0.374 µM, respectively) (4, 12), SFC-1 was not efficiently inhibited by the compounds tested. As expected, no inhibition was observed when the enzyme was assayed with EDTA.

In conclusion, kinetic studies showed that purified SFC-1 exhibits a remarkably broad substrate range, including ß-lactams of all classes, and increased efficiency against ceftazidime. Moreover, this enzyme is less inhibited by class A ß-lactamase inhibitors than other group 2f carbapenemases. Tertiary structures for NMC-A and Sme-1 are already available (7, 9). It would be interesting to generate a crystal structure of the ß-lactamase SFC-1 and investigate the biochemical and structural relationships of SFC-1 in comparison with those of NMC-A and Sme-1.


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ACKNOWLEDGMENTS
 
We are grateful to James Spencer (University of Bristol) for providing meropenem and aztreonam. We thank Merck and Pfizer for the gifts of imipenem and sulbactam, respectively.

This work was supported by Fundação para a Ciência e Tecnologia with grants BD/30490/2006 (F.F.), BPD/7183/2001 (A.C.S.), and BPD/21384/2005 (I.H.). B.S. is a postdoctoral fellow of the Fund for Scientific Research-Flanders (F.W.O.-Vlaanderen, Belgium).


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FOOTNOTES
 
* Corresponding author. Mailing address: CESAM, Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal. Phone: (351) 234 370 783. Fax: (351) 234 426 408. E-mail: antonio.correia{at}ua.pt Back

{triangledown} Published ahead of print on 17 September 2007. Back


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Antimicrobial Agents and Chemotherapy, December 2007, p. 4512-4514, Vol. 51, No. 12
0066-4804/07/$08.00+0     doi:10.1128/AAC.00491-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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