Crystallization and preliminary diffraction studies of SFC-1, a carbapenemase conferring antibiotic resistance

The SFC-1 gene from S. fonticola was cloned and SFC-1 was expressed, purified and crystallized. X-ray diffraction data were collected from an SFC-1 crystal to 1.6 Å resolution.

Abstract

SFC-1, a class A carbapenemase that confers antibiotic resistance, hydrolyzes the β-lactam rings of β-lactam antibiotics (carbapenems, cephalosporins, penicillins and aztreonam). SFC-1 presents an enormous challenge to infection control, particularly in the eradication of Gram-negative pathogens. As SFC-1 exhibits a remarkably broad substrate range, including β-lactams of all classes, the enzyme is a potential target for the development of antimicrobial agents against pathogens producing carbapenemases. In this study, SFC-1 was cloned, overexpressed, purified and crystallized. The SFC-1 crystal diffracted to 1.6 Å resolution and belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 65.8, b = 68.3, c = 88.8 Å. Two molecules are present in the asymmetric unit, with a corresponding V _M of 1.99 ų Da⁻¹ and a solvent content of 38.1%.

1. Introduction  

Severe clinical problems have arisen from the appearance of antibiotic resistance in pathogenic bacteria that cause nosocomial and/or community-acquired infections (Chopra et al., 2008 ▶; Lee et al., 2009 ▶, 2012 ▶). β-Lactamases hydrolyze the β-lactam rings of β-lactam antibiotics and thus provide pathogenic bacteria with resistance to these antibiotics (Schneider et al., 2011 ▶). Based on their primary structures, β-lactamases have been grouped into four classes (A–D), with classes A, C and D using a serine-based covalent catalysis mechanism (Bush & Jacoby, 2010 ▶; Frère, 1995 ▶). Class A β-lactamases possess a very high degree of sequence diversity (more than 600 variants with 11–66.2% sequence identity to mature SFC-1) and are particularly troublesome in the eradication of Gram-negative pathogens (Bush & Jacoby, 2010 ▶). To overcome inactivation by these β-lactamases, carbapenems such as imipenem and meropenem have been developed (Higgins et al., 2009 ▶). However, class A carbapenemases that can hydrolyze a broad variety of β-lactams (carbapenems, cephalosporins, penicillins and aztreonam) have recently emerged (Papp-Wallace et al., 2011 ▶; Queenan & Bush, 2007 ▶).

To date, five major families of class A carbapenemases have been identified in Gram-negative bacteria: the IMI/NMC-A (imipenemase/non-metallocarbapenemase-A), SME (Serratia marcescens enzyme), KPC (Klebsiella pneumonia carbapenemase), GES (Guiana extended-spectrum) and SFC (S. fonticola carbapenemase) families (Fonseca et al., 2007 ▶; Henriques et al., 2004 ▶; Queenan & Bush, 2007 ▶). Increasing numbers of these β-lactamases are being found in the clinic and the environment, the genes of which are primarily located on plasmids or chromosomes (Henriques et al., 2004 ▶; Queenan & Bush, 2007 ▶). This potential for wide dispersal together with their broad substrate specificity highlights the importance of investigating the structural details of these enzymes. Only four crystal structures of class A carbapenemases have been reported: those of KPC-2 (Ke et al., 2007 ▶), SME-1 (Sougakoff et al., 2002 ▶), GES-2 (Smith et al., 2007 ▶) and NMC-A (Swarén et al., 1998 ▶). SFC-1 has 66.2, 63.4, 62.2 and 33.1% sequence identity to mature KPC-2, NMC-­A, SME-1 and GES-1, respectively. However, the mechanism responsible for carbapenemase activity in class A enzymes remains relatively obscure and the structure of SFC-1 (the only member of the SFC family) has not thus far been reported. Furthermore, SFC-1 exhibited catalytic efficiencies for imipenem and ceftazidime that were higher than those of the structurally known carbapenemases (Fonseca et al., 2007 ▶). Thus, there is a need to determine the crystal structure of SFC-1 in order to understand the biochemical characteristics of class A carbapenemases and to elucidate the structural basis of the expanded catalytic activity of SFC-1 towards carbapenems. Here, we expressed, purified, crystallized and performed a preliminary X-ray crystallo­graphic analysis of SFC-1.

2. Materials and methods  

2.1. Cloning  

The class A carbapenemase SFC-1 was produced by S. fonticola UTAD54 (Henriques et al., 2004 ▶). To express the SFC-1 gene (bla _SFC-1) as a soluble form in Escherichia coli, the bla _SFC-1 gene with codon optimization was chemically synthesized using GenScript technology (GenScript Corp., Piscataway, New Jersey, USA) and amplified by polymerase chain reaction (PCR). The sequences of the forward and reverse oligonucleotide primers designed from the bla _SFC-1 gene for PCR were as follows: 5′-ATA CAT ATG CAC CAT CAT CAT CAT CAT GAC GAC GAC GAC AAG AAT ATG GCC GAA GCT GCC T-3′ (NdeI restriction site in bold) and 5′-GAG CTC GAG TCA GAA ACC GAT TGA TTT ACC C-3′ (XhoI restriction site in bold), respectively. The underlined bases indicate the hexahistidine-tag site and the italic bases indicate the enterokinase recognition site. The amplified gene was double-digested with NdeI and XhoI and inserted into the expression vector pET-30a (Novagen, Madison, Wisconsin, USA) digested with the same DNA-restriction enzymes to produce the pET-30a/His₆-bla _SFC-1 plasmid. After verifying the DNA sequence, the plasmid DNA was transformed into E. coli strain BL21 (DE3) for overexpression of His₆-SFC-1 (Fig. 1 ▶).

Figure 1.

Schematic diagram of the recombinant protein His₆-SFC-1 including the first residue (methionine, M), the His₆ tag, the enterokinase recognition site (DDDDK) and mature SFC-1 (residues 22–309) without the signal peptide (residues 1–21; MSRTGRLSVFFSAIFPLLTLT). The fused peptide (M, His₆ tag and enterokinase recognition site) was removed from the recombinant protein during the purification process and the purified mature SFC-1 (residues 22–309) was crystallized. 1, 26, 27 and 309 are residue numbers of immature SFC-1 (residues 1–309).

2.2. Overexpression and purification  

The transformed cells were grown in Luria–Bertani medium (Difco, Detroit, Michigan, USA) containing 50 µg ml⁻¹ kanamycin to an OD₆₀₀ of 0.5 at 310 K. The expression of His₆-SFC-1 was induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 16 h at 289 K. The cells were harvested by centrifugation at 3000g (Supra 30K A1000S-4 rotor; Hanil, Seoul, Republic of Korea) for 30 min at 277 K. The resulting cell pellet was resuspended in ice-cold lysis buffer (10 mM Tris–HCl pH 7.0) and homogenized with a sonicator (Sibata, Saitama, Japan). The crude lysate was centrifuged at 19 960g (Hanil) for 30 min at 277 K and the clarified supernatant was loaded onto a His-Bind column (Novagen, Wisconsin, USA) equilibrated with binding buffer (20 mM Tris–HCl, 10 mM imidazole, 500 mM NaCl pH 7.9). His₆-SFC-1 was eluted with the same buffer containing 1 M imidazole. Eluted fractions containing His₆-SFC-1 were pooled and concentrated to a volume of approximately 1 ml using Vivaspin 20 concentrators (Sartorius, Göttingen, Germany). For further purification, the His₆ tag was removed from His₆-SFC-1 using entero­kinase according to the manufacturer’s instructions (Roche Molecular Biochemicals, Mannheim, Germany). The reaction mixture was desalted and concentrated using a Fast Desalting column (GE Healthcare Biosciences AB, Uppsala, Sweden) and then loaded onto a Superdex 200 prep-grade column (GE Healthcare) previously equilibrated with 10 mM MES pH 6.8, 5 mM dithiothreitol for further purification by gel filtration. The homogeneity of the purified protein was analyzed via SDS–PAGE (Fig. 2 ▶ a). The purified SFC-1 without the His₆ tag was dialyzed against 10 mM MES pH 6.8 and subsequently concentrated to 7 mg ml⁻¹ for crystallization trials.

Figure 2.

Purified SFC-1 and its crystals. (a) Purified SFC-1 shown on 12% SDS–PAGE. The left lane contains molecular-mass markers (labelled in kDa). (b) Crystals of SFC-1 with approximate dimensions of 0.2 × 0.1 × 0.05 mm.

2.3. Crystallization and X-ray data collection  

Crystallization conditions were screened by the sitting-drop vapour-diffusion method using a Hydra II eDrop automated pipetting system (Matrix) at 287 K. Drops consisted of 0.5 µl protein solution (7 mg ml⁻¹) and 0.5 µl reservoir solution and were equilibrated against 70 µl reservoir solution at 287 K. The initial crystallization conditions tested were from the Index kit (Hampton Research). After two weeks, crystals were observed in Index condition No. 44 [0.1 M HEPES pH 7.5, 25%(w/v) PEG 3350]. Crystals were cryoprotected in the reservoir solution supplemented with 20%(v/v) PEG 400. The crystal was mounted in a loop and transferred into the cryoprotectant solution for 1 min prior to cooling in liquid nitrogen. The cryoprotected crystal was then mounted on the goniometer in a stream of cold nitrogen at 100 K. X-ray diffraction data were collected using an ADSC Q315r detector on beamline 5C SB II at Pohang Light Source (PLS), Republic of Korea. X-ray diffraction data to 1.6 Å resolution were collected from the SFC-1 crystal. The crystal was oscillated by 1.0° per frame over a range of 360° to obtain maximum redundancy. All data were integrated and scaled using the DENZO and SCALEPACK crystallographic data-reduction routines (Otwinowski & Minor, 1997 ▶).

3. Results and discussion  

Crystals were obtained by mixing 0.5 µl protein solution with 0.5 µl reservoir solution consisting of 0.1 M HEPES pH 7.5, 25%(w/v) PEG 3350. After three weeks, crystals grew to maximum dimensions of approximately 0.2 × 0.1 × 0.05 mm (Fig. 2 ▶ b). X-ray diffraction data were collected on beamline 5C SB II at PLS. Data-collection and processing statistics are given in Table 1 ▶. Auto-indexing was performed with DENZO and the results indicated that the crystal belonged to space group P2₁2₁2₁, with unit-cell parameters a = 65.8, b = 68.3, c = 88.8 Å. For space group P2₁2₁2₁ two protomers exist in the asymmetric unit, with a corresponding V _M of 1.99 ų Da⁻¹ and a solvent content of 38.1%. Self-rotation functions were calculated at χ = 180, 120, 90 and 60° to detect twofold, threefold, fourfold and sixfold rotation axes, respectively (Fig. 3 ▶). Only twofold rotation axes were observed, as expected for this space group. The MOLREP program was used for molecular replacement using the class A carbapenemase KPC-2 from K. oxytoca (PDB entry 2ov5; 66.2% sequence identity; Ke et al., 2007 ▶) as a search model. The molecular-replacement solution provided informative 2F _o − F _c and F _o − F _c electron-density maps for model improvement. The two protomers in the asymmetric unit were related by twofold noncrystallographic symmetry (NCS) and the twofold NCS axis was parallel to the a axis. The structural details will be described in a separate paper.

Table 1. Data-collection statistics.

Values in parentheses are for the highest resolution shell.

Beamline	5C SB II, PAL
Wavelength (Å)	0.97951
Resolution range (Å)	50.00–1.60 (1.63–1.60)
Space group	P212121
Unit-cell parameters (Å)	a = 65.8, b = 68.3, c = 88.8
Total No. of reflections	863384
No. of unique reflections	65381
Completeness (%)	96.8 (73.4)
Molecules per asymmetric unit	2
Solvent content (%)	38.1
Average I/σ(I)	52.5 (4.2)
R merge † (%)	7.1 (27.1)
Multiplicity	13.2 (7.3)

^† R _merge = , where I(hkl) is the intensity of reflection hkl, is the sum over all reflections and is the sum over i measurements of reflection hkl.

Figure 3.

Self-rotation function. (a) χ = 180°, (b) χ = 120°, (c) χ = 90° and (d) χ = 60° sections of the self-rotation function calculated with MOLREP (Vagin & Teplyakov, 2010 ▶) using data from 50 to 3 Å resolution.