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Acta Crystallographica Section F: Structural Biology and Crystallization Communications logoLink to Acta Crystallographica Section F: Structural Biology and Crystallization Communications
. 2009 Dec 25;66(Pt 1):37–40. doi: 10.1107/S1744309109047708

Crystallization and preliminary X-ray diffraction analysis of diaminopimelate epimerase from Escherichia coli

Lilian Hor a,b,c,‡, Renwick C J Dobson b,c,d,‡, Con Dogovski b,c, Craig A Hutton a,c, Matthew A Perugini b,c,*
PMCID: PMC2805532  PMID: 20057066

Diaminopimelate (DAP) epimerase, an enzyme in the lysine-biosynthetic pathway, is a promising target for antibiotic development against pathogenic bacteria. Here, the cloning, expression, purification, crystallization and preliminary diffraction analysis of DAP epimerase from E. coli are reported.

Keywords: antibiotics, antibiotic resistance, cell-wall synthesis, diaminopimelate epimerase, drug discovery, Escherichia coli, lysine biosynthesis

Abstract

Diaminopimelate (DAP) epimerase (EC 5.1.1.7) catalyzes the penultimate step of lysine biosynthesis in bacteria and plants, converting l,l-diaminopimelate to meso-diaminopimelate. Here, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DAP epimerase from Escherichia coli are presented. Crystals were obtained in space group P41212 and diffracted to 2.0 Å resolution, with unit-cell parameters a = b = 89.4, c = 179.6 Å. Molecular replacement was conducted using Bacillus anthracis DAP epimerase as a search model and showed the presence of two molecules in the asymmetric unit, with an initial R free of 0.456 and R work of 0.416.

1. Introduction

Diaminopimelate (DAP) epimerase (EC 5.1.1.7) is a member of the PLP-independent amino-acid racemases. It catalyzes the stereoinversion of l,l-diaminopimelate to meso-diaminopimelate in the lysine-biosynthetic pathway in plants and bacteria. Since lysine bio­synthesis does not occur in animals, the members of the pathway, such as DAP epimerase, are attractive targets for rational antibiotic design (Hutton et al., 2003 ▶, 2007 ▶).

The catalytic mechanism of DAP epimerase has been extensively studied, showing that the enzyme utilizes a ‘two-base’ mechanism involving a pair of cysteine residues (Pillai et al., 2006 ▶; Wiseman & Nichols, 1984 ▶). One residue acts as a base and the other as an acid, resulting in an inversion of stereochemical configuration.

The structures of DAP epimerases from a variety of organisms have been determined, including those from Haemophilus influenzae (Cirilli et al., 1998 ▶; Lloyd et al., 2004 ▶), Mycobacterium tuberculosis (Usha et al., 2009 ▶) and the plant Arabidopsis thaliana (Pillai et al., 2009 ▶). All of the structures show a two-domain α/β-fold, with each domain contributing one active-site cysteine. The enzyme is a symmetrical monomer comprised of two domains, each containing eight β-strands and two α-helices. This unique fold was first observed in H. influenzae DAP epimerase; since then, a number of proteins including a phenazine-biosynthetic protein (Blankenfeldt et al., 2004 ▶; Parsons et al., 2004 ▶), a proline racemase (Buschiazzo et al., 2006 ▶) and a methyl­aconitate isomerase (Garvey et al., 2007 ▶) have also been shown to adopt this fold.

Here, we present the cloning, recombinant expression, purification, crystallization and preliminary crystallographic studies of DAP epimerase from Escherichia coli.

2. Materials and methods

2.1. Cloning, expression and purification of E. coli DAP epimerase

The dapF gene encoding DAP epimerase, including flanking sequences, was amplified by PCR from E. coli genomic DNA using primers 5′-GTCCATCCCAGGTGAATTAC-3′ and 5′-GTGAGTG­TTTCCTGCAGTTC-3′. The PCR product was subsequently cloned into pCR-Blunt II-TOPO (Invitrogen) using the Zero Blunt TOPO PCR Cloning Kit (Invitrogen). NdeI and BamHI restriction sites were incorporated into the 5′ and 3′ ends of the dapF gene by PCR using the primers 5′-CATATGCAGTTCTCGAAAATGC-3′ and 5′-GGATCCTCAATGGTGATGGTGATGGTGTAGATGAATAAA­TCCGTCGT-3′, respectively. The 3′-end primer was also designed to append a carboxy-terminal hexahistidine tag to the dapF open reading frame. The resulting PCR product was digested with the restriction enzymes NdeI and BamHI and subsequently cloned into the corresponding sites of the pET11a expression vector (New England Biolabs). The identity of the E. coli dapF gene was verified by restriction analysis and dideoxynucleotide sequencing.

E. coli DAP epimerase was expressed in E. coli BL21 (DE3) harbouring the pET11a-dapF plasmid by culturing at 293 K in auto-induction media (Studier, 2005 ▶) containing 100 µg ml−1 ampicillin for 25 h. The cells were harvested by centrifugation at 5000g for 15 min at 277 K and the cell pellet was stored at 253 K. Cell lysates were prepared as described elsewhere (Burgess et al., 2008 ▶; Dobson et al., 2008 ▶; Voss et al., 2009 ▶). Briefly, harvested cells were resuspended in 20 mM Tris, 500 mM NaCl, 20 mM imidazole, 5 mM DTT pH 8.0 and lysed by sonication and the supernatant was collected by centrifugation at 30 000g for 20 min at 277 K. DAP epimerase was then purified at 277 K from the cell lysate by immobilized metal-affinity chromatography using 3 × 5 ml HisTrap HP columns connected in series (GE Healthcare) pre-equilibrated with 20 mM Tris, 500 mM NaCl, 20 mM imidazole, 1 mM DTT pH 8.0. His-tagged DAP epimerase was then eluted with a 20–500 mM imidazole gradient over six column volumes. Fractions were subsequently analysed by SDS–PAGE and those containing pure DAP epimerase were pooled and dialyzed into 20 mM Tris, 1 mM DTT pH 7.8. The enzyme was then concentrated to 12 mg ml−1 and further purified by gel-filtration chromatography using a 1.0 cm (diameter) × 30 cm (length) Sephacryl S-200 column (GE Healthcare) pre-equilibrated in 20 mM Tris, 1 mM DTT pH 7.8. For crystallization trials, the recombinant protein was concentrated to 8.0 mg ml−1.

2.2. Crystallization of E. coli DAP epimerase

Crystallization screens were conducted as described previously (Atkinson et al., 2009 ▶; Burgess et al., 2008 ▶; Dobson et al., 2008 ▶; Voss et al., 2009 ▶). Initial protein-crystallization experiments were performed at the CSIRO node of the Bio21 Collaborative Crystallization Centre (C3) using the PACT Suite and JCSG+ Suite crystal screens at 281 and 293 K (Newman et al., 2005 ▶, 2008 ▶). The screens were set up using the sitting-drop vapour-diffusion method with droplets consisting of 150 nl protein solution and 150 nl reservoir solution. Conditions that yielded crystals were replicated using the hanging-drop vapour-diffusion method with drops containing 2 µl protein solution and 2 µl precipitant solution at 281 and 293 K. These conditions were also trialled with the addition of 5 mM TCEP. The addition of TCEP yielded crystals with different morphologies to those obtained with DTT alone. The crystal shown in Fig. 1 ▶ was obtained at 293 K from a 4 µl drop formed from 2 µl protein solution (8.0 mg ml−1 in 20 mM Tris, 5 mM DTT, 5 mM TCEP pH 7.8) and 2 µl precipitant [200 mM sodium nitrate, 20%(w/v) polyethylene glycol 3350, 100 mM bis-tris propane pH 8.5]. We noted that only freshly purified protein readily produced crystals.

Figure 1.

Figure 1

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Crystal of E. coli DAP epimerase. The bar represents 150 µm.

2.3. Data collection and processing

For X-ray data collection, the crystal was soaked in a cryoprotectant solution consisting of reservoir solution made up with 20%(v/v) glycerol and directly flash-cooled in liquid nitrogen. Intensity data were collected at 110 K at the Australian Synchrotron (MX2 beamline). Data were collected in 0.5° oscillations over 95° using an ADSC Q315r image-plate detector positioned 230 mm from the crystal, with an exposure time of 0.5 s and 95% attenuation. The diffraction data were processed using the programs MOSFLM (Leslie, 1991 ▶) and SCALA (Collaborative Computational Project, Number 4, 1994 ▶). Although 90° of data were initially collected, the resolution and quality of the diffraction data decreased (as judged by an increased batch R merge and number of rejected reflections); as such, the final 34° wedge of data was omitted from subsequent steps.

3. Results and discussion

E. coli DAP epimerase was cloned, expressed and purified to homogeneity by a two-step purification protocol involving Ni2+-affinity chromatography and gel-filtration chromatography. Fractions following each step of the purification strategy were assessed by SDS–PAGE (Fig. 2 ▶) and the enzyme activity was measured using the DAP epimerase–DAP dehydrogenase coupled assay (Cox et al., 2002 ▶). Gel-filtration chromatographic analysis indicated evidence of large-molecular-mass species that were believed to be aggregated recombinant DAP epimerase. The aggregate did not display enzymatic activity. Removal of this incompetent enzyme resulted in a twofold increase in the specific activity (Table 1 ▶).

Figure 2.

Figure 2

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SDS–PAGE analysis of E. coli DAP epimerase (molecular mass 31 031.56 Da) at each stage of the purification. Lane 1, molecular-mass markers (kDa); lane 2, cell lysate pre-induction; lane 3, cell lysate post-induction; lane 4, after Ni2+-affinity liquid chromatography; lane 5, after gel-filtration liquid chromatography. The SDS–PAGE gel (12%) was stained with Coomassie Brilliant Blue R250.

Table 1. Purification of E. coli DAP epimerase.

One unit of enzyme activity is equal to the reduction of 1 µmol of NADP+ per second.

  Total protein (mg) Total activity (U) Specific activity (U mg−1) Degree of purification (fold)
Crude extract 504 103 0.205  
Ni2+-affinity liquid chromatography 98.7 56.7 0.574 2.80
Gel-filtration chromatography 39.2 46.1 1.18 5.76
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E. coli DAP epimerase was found to crystallize in a number of conditions from the PACT screen, in particular those containing PEG 3350 and bis-tris propane buffer. The crystal in Fig. 1 ▶ appeared after 2 d and continued to grow to a length of 150 µm in the longest dimension over a further 5 d.

Data were collected at the Australian Synchrotron (Melbourne, Australia) using the micro-focus beamline MX2. This allowed only one end of the crystal to be exposed to X-rays in order to prevent diffraction from the cluster of crystals that can be observed in the midsection of the main crystal in Fig. 1 ▶. The crystal showed excellent diffraction (to beyond 2.0 Å resolution; Fig. 3 ▶) and belonged to space group P41212, with unit-cell parameters a = b = 89.4, c = 179.6 Å. R merge was 0.077, R p.i.m. was 0.039, R r.i.m was 0.087, V M was 2.89 Å3 Da−1 and the estimated solvent content was 57% assuming the presence of two molecules in the asymmetric unit. Complete data-collection statistics are summarized in Table 2 ▶.

Figure 3.

Figure 3

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An X-ray diffraction frame of the cryocooled crystal of E. coli DAP epimerase. The crystal diffracted to beyond 2.0 Å (corners).

Table 2. X-ray data-collection statistics.

Values in parentheses are for the highest resolution shell. The Matthews coefficient and solvent content are based on two monomers of molecular weight 31 031.56 Da in the asymmetric unit (ASU).

Wavelength (Å) 0.97946
No. of images 111
Oscillation (°) 0.5
Space group P41212
Unit-cell parameters (Å) a = b = 89.4, c = 179.6
Resolution (Å) 44.69–2.00 (2.11–2.00)
Observed reflections 215760 (29012)
Unique reflections 47663 (6683)
Completeness (%) 96.1 (93.7)
Rmerge† 0.077 (0.339)
Rp.i.m.‡ 0.039 (0.137)
Rr.i.m. 0.087 (0.385)
Mean I/σ(I) 13.0 (4.2)
Redundancy 4.5 (4.3)
Wilson B value (Å2) 23.4
Molecules per ASU 2
VM (Å3 Da−1) 2.89
Solvent content (%) 57
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†

R merge = Inline graphic Inline graphic.

‡

R p.i.m. = Inline graphic Inline graphic Inline graphic.

Molecular replacement using DAP epimerase from Bacillus anthracis as a search model (PDB code 2otn; 37% sequence identity) was performed using Phaser (McCoy et al., 2007 ▶). This confirmed the presence of two molecules in the asymmetric unit, with an initial R free of 0.456 and R work of 0.416. Further model building and refinement continues.

Acknowledgments

We would firstly like to acknowledge the support and assistance of the friendly staff, especially Dr Janet Newman, at the Bio21 Collaborative Crystallographic Centre at CSIRO Molecular and Health Technologies, Parkville, Melbourne as well as all members of the Perugini and Hutton laboratories for helpful discussions during the preparation of this manuscript. This research was undertaken on the MX2 beamline at the Australian Synchrotron, Victoria, Australia. The views expressed herein are those of the authors and are not necessarily those of the owner or operator of the Australian Synchrotron. Finally, we acknowledge the Defense Threat Reduction Agency (DTRA; Project ID AB07CBT004) for program funding, the Australian Research Council for providing a Future Fellowship to MAP and The University of Melbourne for the C. R. Roper Research Fellowhip to RCJD.

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