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. 2012 Aug 30;68(Pt 9):1040–1047. doi: 10.1107/S1744309112033052

Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from Agrobacterium tumefaciens

Sarah C Atkinson a,b, Con Dogovski a, Renwick C J Dobson b,c, Matthew A Perugini a,b,*
PMCID: PMC3433193  PMID: 22949190

Dihydrodipicolinate synthase from the plant pathogen A. tumefaciens has been cloned, expressed, purified and crystallized in its unliganded form, in the presence of its substrate pyruvate and in the presence of pyruvate and the allosteric inhibitor lysine. Diffraction data for the crystals were collected to a maximum resolution of 1.40 Å.

Keywords: crown gall disease, herbicides, lysine metabolism, quaternary structure

Abstract

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the lysine-biosynthesis pathway in bacteria, plants and some fungi. This study describes the cloning, expression, purification and crystallization of DHDPS (NP_354047.1) from the plant pathogen Agrobacterium tumefaciens (AgT-DHDPS). Enzyme-kinetics studies demonstrate that AgT-DHDPS possesses DHDPS activity in vitro. Crystals of AgT-DHDPS were grown in the unliganded form and in forms with substrate bound and with substrate plus allosteric inhibitor (lysine) bound. X-ray diffraction data sets were subsequently collected to a maximum resolution of 1.40 Å. Determination of the structure with and without substrate and inhibitor will offer insight into the design of novel pesticide agents.

1. Introduction  

Crown gall disease is a significant plant disease that affects a large range of agriculturally important species, including deciduous fruits, vines and vegetables (De Cleene & De Ley, 1976 ▶). The aetiological agent of the disease, the bacterium Agrobacterium tumefaciens (AgT), infects healthy plants and causes crop decline or mortality (Agrios, 2004 ▶; Burr et al., 1998 ▶). The disease is of genuine concern because of its worldwide distribution and its potential to cause considerable damage to the agricultural industry. At risk are several important commodities including grapes, the most cultivated fruit in the world (Giribaldi & Giuffrida, 2010 ▶), and stone fruits (Kerr, 1969 ▶). Therefore, it is surprising that there is still no universally effective treatment for crown gall disease (Anand et al., 2008 ▶). Existing strategies for pest control have significant limitations (Burr & Otten, 1999 ▶; Escobar et al., 2001 ▶; Lee et al., 2003 ▶; Viss et al., 2003 ▶) and it is clear that there is a real need to develop novel pesticides targeting essential metabolic processes for the treatment of this disease. One such target is the lysine-biosynthesis pathway in A. tumefaciens.

Lysine biosynthesis occurs in bacteria, plants and some fungi (Hutton et al., 2007 ▶; Dogovski et al., 2009 ▶, 2012 ▶). The lysine-biosynthesis pathway yields lysine, which is essential for protein synthesis, and meso-DAP, which is a vital constituent of the peptido­;glycan layer of the bacterial cell wall (Hutton et al., 2007 ▶; Dogovski et al., 2009 ▶, 2012 ▶). The pathway is thus of interest for the development of novel antimicrobial agents, given that several enzymes that function in lysine biosynthesis are essential to bacteria (Hutton et al., 2007 ▶; Dogovski et al., 2009 ▶). These essential enzymes include dihydro­dipicolinate synthase (DHDPS), which catalyses the first committed step in the pathway (Mirwaldt et al., 1995 ▶; Dobson et al., 2005 ▶; Perugini et al., 2005 ▶; Burgess et al., 2008 ▶; Dogovski et al., 2009 ▶, 2012 ▶; Voss et al., 2010 ▶). DHDPS has been shown to be one of only 271 essential genes in Bacillus subtilis (Kobayashi et al., 2003 ▶) and is thus considered to be an attractive target for rational drug design (Hutton et al., 2003 ▶, 2007 ▶; Mitsakos et al., 2011 ▶). However, no potent inhibitors have been identified to date. Interestingly, several compounds have displayed clear differentiation in the inhibition of DHDPS enzymes from different species (Mitsakos et al., 2008 ▶), suggesting the potential for targeting the enzyme from specific pathogens. Determination of the crystal structure of DHDPS from A. tumefaciens will thus offer insight into the design of specific crown gall disease inhibitors.

DHDPS catalyzes the condensation of pyruvate and (S)-aspartate semialdehyde [(S)-ASA] to form (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid (HTPA) (Blickling, Renner et al., 1997 ▶; Hutton et al., 2007 ▶; Dogovski et al., 2009 ▶; Fig. 1 ▶). The reaction proceeds via a ping-pong kinetic mechanism in which pyruvate binds as a Schiff base to an active-site lysine residue (Lys162 in AgT-DHDPS; Blickling, Renner et al., 1997 ▶; Dobson, Gerrard et al., 2004 ▶).

Figure 1.

Figure 1

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The reaction catalyzed by DHDPS.

The structures of DHDPS from a number of bacterial species have been determined (Table 1 ▶). Structures of DHDPS from the plant species Nicotiana sylvestris (Blickling, Beisel et al., 1997 ▶), Vitis vinifera (Atkinson et al., 2012 ▶) and Arabidopsis thaliana (Griffin et al., 2012 ▶) have also been described.

Table 1. Examples of bacterial AgT-DHDPS orthologues with known crystal structures.

Organism Ligands Identity to AgT-DHDPS (%) Reference
Escherichia coli Unliganded, lysine, pyruvate 44 Mirwaldt et al. (1995 ▶); Dobson et al. (2005 ▶); Devenish et al. (2008 ▶)
Thermotoga maritima Unliganded 38 Pearce et al. (2006 ▶)
Bacillus anthracis Unliganded, pyruvate 40 Blagova et al. (2006 ▶); Voss et al. (2010 ▶)
Mycobacterium tuberculosis Unliganded 41 Kefala et al. (2008 ▶)
Hahella chejuensis Unliganded 57 Kang et al. (2010 ▶)
Staphylococcus aureus Unliganded, pyruvate 40 Burgess et al. (2008 ▶); Girish et al. (2008 ▶)
Corynebacterium glutamicum Unliganded 39 Rice et al. (2008 ▶)
Neisseria meningitidis Unliganded 45 Devenish et al. (2009 ▶)
Pseudomonas aeruginosa Unliganded, lysine 45 Kaur et al. (2011 ▶)
Methanococcus jannaschii Unliganded 46 Padmanabhan et al. (2009 ▶)
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In bacteria, the enzyme is typically comprised of a homotetramer described as a ‘head-to-head’ dimer of dimers, as depicted in Fig. 2 ▶(a). Each monomer adopts a (β/α)8 or TIM-barrel fold. Both the active and allosteric sites are located at the so-called ‘tight-dimer interface’, with the allosteric cleft, which binds lysine, at the top and bottom of the tetramer (Dobson et al., 2005 ▶; Fig. 2 ▶ a). More recently, studies of DHDPS from a number of species have shown that the enzyme can adopt alternative oligomeric states and quaternary architectures. For instance, DHDPS from Pseudomonas aeruginosa and Staphylococcus aureus exists as dimers (Burgess et al., 2008 ▶; Girish et al., 2008 ▶; Kaur et al., 2011 ▶). Although the enzymes from plant species are homotetrameric (Blickling, Beisel et al., 1997 ▶; Atkinson et al., 2012 ▶; Griffin et al., 2012 ▶), they adopt an alternative ‘back-to-back’ quaternary architecture (Fig. 2 ▶ b). Of interest to this study, there are three structures in the PDB of putative DHDPS enzymes from A. tumefaciens that adopt noncanonical quaternary forms, namely dimers [PDB entries 2r8w (NP_353919.2; Fig. 2 ▶ c) and 3b4u (NP_355449.1; Fig. 2d)] and a hexamer [PDB entry 2hmc (NP_355989.1; Fig. 2 ▶ e)] (Midwest Center for Structural Genomics, unpublished work). In addition, comparative sequence analyses of 2r8w, 3b4u and 2hmc show that all three structures lack one or more of the key active-site residues shown to be important for DHDPS catalytic function (Fig. 3 ▶). However, the product of the putative dapA gene from A. tumefaciens (NP_354047; designated here as AgT-DHDPS) contains all of the key catalytic residues and also shows high conservation of the allosteric site residues, suggesting that this protein functions as a DHDPS enzyme (Fig. 3 ▶). This study describes the cloning, expression, purification, crystallization and preliminary X-ray diffraction analyses of a promising antimicrobial target from A. tumefaciens, namely DHDPS. Determination of the structure of AgT-DHDPS will thus offer insight into the design of novel pesticide agents for the treatment of crown gall disease.

Figure 2.

Figure 2

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(a) DHDPS from E. coli (PDB entry 1dhp; Mirwaldt et al., 1995 ▶) and (b) DHDPS from V. vinifera (PDB entry 3tuu; Atkinson et al., 2012 ▶), with the location of the active site marked. (c–e) DHDPS from A. tumefaciens: (c) PDB entry 2r8w, (d) PDB entry 3b4u, (e) PDB entry 2hmc (Midwest Center for Structural Genomics, unpublished work).

Figure 3.

Figure 3

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Sequence alignment of E. coli DHDPS and ten putative dapA gene products in the annotated A. tumefaciens strain C58 genome. Residues of the active site (green), including the catalytic triad (yellow) and the catalytic lysine (red), are highlighted.

2. Materials and methods  

2.1. Construction of an AgT-DHDPS expression vector  

The gene encoding AgT-DHDPS (NP_35047.1) was amplified by PCR from genomic DNA derived from A. tumefaciens strain C58 (forward primer 5′-GACTGTCACACTTCCGACAA-3′ and reverse primer 5′-TCTCGCTTGTTCAACAGCAG-3′). Amplified DNA was cloned into pCR-Blunt II-TOPO (Invitrogen) to produce the vector pTOPO-NP_354047.1. The presence of the gene encoding AgT-DHDPS was confirmed by restriction-endonuclease analysis and dideoxynucleotide sequencing. The forward and reverse primers 5′-GACGACGACAAGATGTTCAAGGGATCAATTCCCGC-3′ and 5′-GAGGAGAAGCCCGGTTCAGTTCATCAGGCCGGC­ATG-3′, respectively, facilitated PCR amplification and subcloning of the gene into the expression vector pET-46 Ek/LIC (Novagen). The sequence of the resulting vector (pET-His-NP_354047.1) was confirmed via dideoxynucleotide sequencing and allows expression of AgT-DHDPS with an amino-terminal hexahistidine tag (MAHHHHHHVDDD­DEK).

2.2. Expression and purification of AgT-DHDPS  

Escherichia coli BL21 (DE3) cells were transformed with pET-His-NP_354047.1 and plated onto Luria–Bertani (LB) agar containing 25 µg ml−1 kanamycin. Following overnight incubation at 310 K, single colonies were transferred into 10 ml LB broth containing 25 µg ml−1 kanamycin and grown overnight at 310 K with shaking (180 rev min−1). The cells were diluted 1/100 in flasks containing 1 l LB broth (25 µg ml−1 kanamycin) and cultivated at 303 K with continuous shaking (180 rev min−1) until an OD600 nm of 0.6 was attained. The cells were subsequently treated with 1.0 mM isopropyl β-d-1-thiogalactopyranoside to induce the expression of recombinant AgT-DHDPS. The cultures were incubated for a further 3 h at 303 K with continuous shaking (180 rev min−1). The cells were immediately harvested by centrifugation at 16 000g for 20 min at 277 K.

The cell pellet was resuspended in 30 ml buffer I (20 mM Tris, 500 mM NaCl, 20 mM imidazole pH 8.0) and the cells were lysed by sonication using an MSE Soniprep 150 sonicator with an 18 mm diameter titanium probe at a power output of 10–14 µm. Cellular debris was pelleted by centrifugation (30 min, 48 000g) and the soluble fraction was filtered using a 0.45 µm filter (Millipore) and applied onto a 5 ml His-Trap HP column (GE Healthcare) pre-equilibrated with three column volumes of buffer I. The column was washed in buffer I until a steady baseline absorbance (A 280 nm) was observed. AgT-DHDPS was eluted via a gradient of 100–0% buffer I/0–100% buffer II (20 mM Tris, 500 mM NaCl, 500 mM imidazole pH 8.0) applied over ten column volumes. AgT-DHDPS eluted at an imidazole concentration of approximately 200 mM.

The eluate was dialysed overnight against buffer III (20 mM Tris, 150 mM NaCl pH 8.0) before storage at 193 K. Prior to crystallization, the protein was thawed overnight at 277 K before size-exclusion liquid chromatography using an XK 16/20 column packed with Superdex 200 resin (30 ml bed volume; GE Healthcare) pre-equilibrated with buffer III. If required, the protein solution was concentrated to approximately 10 mg ml−1 using a 10 kDa cutoff Centricon (Millipore) prior to gel-filtration chromatography. All purification steps were carried out at 277 K. The results of each purification step are shown in Fig. 4 ▶. Fractions following liquid chromatography were also assessed for DHDPS enzymatic activity using the ο-aminobenzaldehyde assay (Yugari & Gilvarg, 1965 ▶), which demonstrated that recombinant AgT-DHDPS possesses DHDPS enzymatic activity (Fig. 5 ▶). For the purification table (Table 2 ▶), kinetic analyses were performed using the DHDPS–DHDPR coupled assay as described previously (Dobson, Gerrard et al., 2004 ▶) using E. coli DHDPR as the coupling enzyme.

Figure 4.

Figure 4

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SDS–PAGE analysis showing the purification of AgT-DHDPS. Lane 1, molecular-weight markers (labelled in kDa); lane 2, non-induced whole cell lysate; lane 3, IPTG-treated whole cell lysate; lane 4, post IMAC chromatography; lane 5, post size-exclusion chromatography.

Figure 5.

Figure 5

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ο-Aminobenzaldehyde assay showing the activity of samples derived from each stage of purification of AgT-DHDPS. Well 1, IPTG-treated whole cell lysate; well 2, post IMAC chromatography; well 3, post size-exclusion chromatography; well 4, negative control (E. coli DHDPR); well 5, positive control (E. coli DHDPS). The purple colour indicates DHDPS activity (Mitsakos et al., 2011 ▶).

Table 2. Purification of recombinant His-tagged AgT-DHDPS.

  Total protein (mg) Total activity (U†) Specific activity (U mg−1) Yield (%) Purification (fold)
Lysate 188 4238 23 — —
IMAC 19 3326 175 78 8
Size exclusion 18 3125 174 74 8
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†

1 U is defined as the consumption of 1 µmol NADPH per minute.

2.3. Crystallization of unliganded and ligand-bound AgT-DHDPS  

Crystallization studies were initially conducted using a 10 mg ml−1 protein preparation of AgT-DHDPS with the full-length hexahistidine tag in 20 mM Tris pH 8.0. Protein crystallization trials were performed at the CSIRO node of the Bio21 Collaborative Crystallization Centre (C3; http://www.csiro.au/c3/) using the PACT Suite and JCSG+ Suite crystal screens (Qiagen; Newman et al., 2005 ▶) at 281 and 293 K as described previously (Dobson, Atkinson et al., 2008 ▶; Dommaraju et al., 2010 ▶; Hor et al., 2010 ▶; Sibarani et al., 2010 ▶; Wubben et al., 2010 ▶; Atkinson et al., 2011 ▶). Screens were performed by the sitting-drop vapour-diffusion method with droplets consisting of 150 nl protein solution (10 mg ml−1) and 150 nl reservoir solution. Several screen conditions produced crystals with varying morphology at 293 K. In addition, trials with Crystal Screen, Crystal Screen 2 and Crystal Screen Cryo (Hampton Research) were also performed in-house using the hanging-drop vapour-diffusion method. 2 µl protein solution and 2 µl precipitant solution were equilibrated against 1 ml reservoir solution in 24-well Linbro plates at 293 K. Optimization of the initial hits by varying the pH and the concentration of the precipitant was carried out as for the Hampton Research screens in 24-well Linbro plates. The best diffracting crystals grew overnight in 0.1 M Tris pH 8.5, 2 M ammonium sulfate. Pyruvate cocrystals obtained using 20 mM pyruvate (1 µl 1 M pyruvate added to 49 µl protein solution just prior to setup of crystal trays) also grew in this condition. The Hampton Research screens described above were also used to obtain lysine and pyruvate cocrystals (1 µl 1 M pyruvate and 1 µl 1 M lysine added to 48 µl protein solution just prior to setup of crystal trays), with the best crystals growing in 2 d from a reservoir solution consisting of 0.17 M lithium sulfate monohydrate, 0.085 M Tris pH 8.5, 25.5%(w/v) PEG 4000, 15%(v/v) glycerol.

2.4. Data collection and processing  

For each condition, diffraction data were collected using a single crystal (Fig. 6 ▶) on either the MX1 or the MX2 beamline (Evans & Pettifer, 2001 ▶; McPhillips et al., 2002 ▶) at the Australian Synchrotron. MX1 uses a 03BM1 (dipole/bending-magnet) source and an ADSC Q210r CCD detector. MX2 uses a 03ID1 (3 m in-vacuum undulator) source and an ADSC Q315r CCD detector. Crystals that had been flash-cooled in liquid nitrogen were mounted on the beamline in a cold nitrogen stream at 110 K. For the unliganded crystal, data were collected on the MX2 beamline in 0.5° steps for one 360° pass with 90% attenuation and an exposure time of 2 s with the detector positioned 160 mm from the crystal. The pyruvate crystal was mounted on the MX1 beamline at 160 mm from the detector and data were collected in 0.5° steps for one 360° pass with an exposure time of 2 s. The lysine/pyruvate crystal was mounted on the MX2 beamline at 140 mm from the detector and data were collected in 1° steps for one 220° pass with 80% attenuation and an exposure time of 2 s. Indexing and integration of the data was performed using MOSFLM (Leslie & Powell, 2007 ▶). POINTLESS (Evans, 2006 ▶) from the CCP4 program suite (Winn et al., 2011 ▶) was run to verify the space group, and scaling and data reduction were performed using SCALA (Evans, 2006 ▶), also from CCP4. All relevant data-collection and processing parameters are given in Table 3 ▶. Data were processed to a resolution at which I/σ(I) was >2 but with reasonable values for both R merge and completeness. Images will be made available via the TARDIS server (Androulakis et al., 2008 ▶) once the structures have been solved and published.

Figure 6.

Figure 6

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Crystals of recombinant AgT-DHDPS: (a) unliganded form, (b) cocrystallized with 20 mM pyruvate and (c) cocrystallized with 20 mM lysine and 20 mM pyruvate. The bar in each image indicates 100 µm.

Table 3. Data-collection and processing statistics.

Values in parentheses are for the highest resolution bin.

  AgT-DHDPS AgT-DHDPS + pyruvate AgT-DHDPS + pyruvate and lysine
Wavelength (Å) 0.9536 0.9536 0.9536
No. of images 673 720 212
Step range (°) 0.5 0.5 1
Space group P212121 C2 C2221
Unit-cell parameters (Å, °) a = 94.8, b = 131.7, c = 162.6 a = 182.8, b = 77.0, c = 98.0, β = 121.2 a = 89.1, b = 122.8, c = 129.6
Resolution (Å) 38.70–1.55 (1.63–1.55) 39.10–1.45 (1.51–1.43) 26.37–1.40 (1.48–1.40)
Observed reflections 2812538 (169697) 1491073 (162951) 457924 (39292)
Unique reflections 265747 (38083) 207498 (25640) 135072 (17665)
Completeness (%) 98.4 (89.9) 96.0 (81.6) 97.2 (88.1)
R merge † 0.077 (0.415) 0.070 (0.344) 0.044 (0.289)
R r.i.m. ‡ 0.081 (0.470) 0.075 (0.374) 0.052 (0.359)
R p.i.m. § 0.023 (0.212) 0.028 (0.144) 0.026 (0.207)
Mean I/σ(I) 17.9 (2.9) 16.5 (4.7) 15.8 (3.1)
Multiplicity 9.7 (4.5) 7.2 (6.4) 3.4 (2.2)
Wilson B value (Å2) 15.4 11.7 10.6
Matthews coefficient (Å3 Da−1) 3.84 2.19 2.73
Molecules per asymmetric unit 4 4 2
Solvent content (%) 68 44 54
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†

Inline graphic Inline graphic.

‡

Inline graphic Inline graphic Inline graphic.

§

R p.i.m. = Inline graphic Inline graphic. Ii(hkl) is the ith intensity measurement of reflection hkl, 〈I(hkl)〉 is its average and N is the redundancy of a given reflection.

3. Results and discussion  

A search for dapA genes in the annotated A. tumefaciens strain C58 genome (NCBI references NC_003062.2, NC_003063.2, NC_003064.2 and NC_003065.3) identified ten putative DHDPS sequences (Goodner et al., 2001 ▶; Wood et al., 2001 ▶). A ClustalW2 (Larkin et al., 2007 ▶; Goujon et al., 2010 ▶) alignment of E. coli DHDPS (BAA16355.1) and the ten putative DHDPS sequences was performed (Fig. 3 ▶). Inspection of the primary amino-acid sequences revealed that only one DHDPS sequence (NCBI reference NP_354047.1; AgT-DHDPS) contained all of the key conserved residues known to be essential for catalysis: the catalytic triad (Thr44, Ty107 and Tyr133), Lys161, which forms a Schiff base with pyruvate, Arg138, which is important for (S)-­ASA binding, and Ile203 (Dobson, Valegård et al., 2004 ▶; Dobson et al., 2005 ▶, 2009 ▶; Dobson, Griffin et al., 2008 ▶). In order to study the structure and function of AgT-DHDPS, the gene encoding this protein was cloned as described in §2.1. Significant quantities of soluble recombinant protein were isolated as described in §2.2.

Approximately 18 mg pure protein was obtained from 1 l bacterial cell culture following the two-step purification procedure (Table 2 ▶). The purity of the sample was at least 95% as estimated by SDS–PAGE (Fig. 4 ▶). The ο-aminobenzaldehyde enzyme assay (Yugari & Gilvarg, 1965 ▶) confirmed that the purified recombinant enzyme was active (Fig. 5 ▶). AgT-DHDPS was initially screened for crystallization as described in §2.3. Larger crystals were then grown in-house under optimized conditions selected from the successful high-throughput screening trials (Fig. 6 ▶). Given that cocrystallization yielded diffracting crystals, ligand soaks were not performed. The crystals shown in Fig. 6 ▶ were obtained by mixing 2 µl protein solution (10 mg ml−1) with 2 µl reservoir solution at 293 K.

In preparation for diffraction data collection, unliganded crystals and crystals containing pyruvate were cryoprotected using 10%(v/v) glycerol in reservoir solution (containing the appropriate ligand) and then immediately mounted in a nylon loop and flash-cooled in liquid nitrogen. Crystals containing lysine and/or pyruvate were grown in 15%(v/v) glycerol and thus were directly mounted and cooled. Diffraction data were collected from the unliganded, pyruvate and pyruvate + lysine crystals to resolutions of 1.55, 1.43 and 1.40 Å, respectively (Fig. 7 ▶). The relevant data-collection and processing parameters are provided in Table 3 ▶.

Figure 7.

Figure 7

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X-ray diffraction images from crystals of AgT-DHDPS (Fig. 6 ▶): (a) unliganded, (b) cocrystallized with pyruvate and (c) cocrystallized with lysine and pyruvate.

The unliganded AgT-DHDPS crystal belonged to space group P212121, with unit-cell parameters a = 94.8, b = 131.7, c = 162.6 Å. Based on the deduced molecular weight of 32 833 Da, calculation of the Matthews coefficient suggested the presence of four subunits per asymmetric unit, with an estimated solvent content of 68% (V M = 3.84 Å3 Da−1). Analysis of average Matthews coefficient values for protein structures of molecular weight 20–40 kDa in the PDB showed that a significant proportion have V M values of 3.5 Å3 Da−1 or higher (Kantardjieff & Rupp, 2003 ▶). Scaling and merging of the crystallo­graphic data resulted in an overall R merge of 0.077, with an R merge of 0.350 in the highest resolution shell. The diffraction of the crystal was observed to significantly reduce towards the end of the data set and thus only the first 673 images were processed. No evidence of twinning was observed. Molecular replacement using the program Phaser (McCoy et al., 2007 ▶) employing an E. coli DHDPS monomer (PDB entry 1yxc; Dobson et al., 2005 ▶; 44% identity; Table 1 ▶) as a search model showed an unambiguous solution with four monomers in the asymmetric unit with a translation-function Z-score (TFZ) of 25.9 and a final log-likelihood gain (LLG) of 8379. Given the high solvent content calculated for four monomers, a Phaser search for eight monomers was also performed, but again yielded four monomers in the asymmetric unit.

The crystal from the drop containing pyruvate belonged to space group C2, with four molecules in the asymmetric unit (V M = 2.19 Å3 Da−1). Scaling and merging of the crystallographic data resulted in an overall R merge of 0.069, with an R merge of 0.314 in the highest resolution shell. No evidence of twinning was observed. Molecular replacement employing the unliganded AgT-DHDPS structure as a search model showed an unambiguous solution with four monomers in the asymmetric unit with a translation-function Z-­score (TFZ) of 29.5 and a final log-likelihood gain (LLG) of 14 979.

In contrast, the pyruvate and lysine cocrystal belonged to space group C2221, with two molecules in the asymmetric unit (V M = 2.73 Å3 Da−1). Crystal diffraction significantly decreased at the end of the data set and thus only 212 images were included in processing. Scaling and merging of the crystallographic data resulted in an overall R merge of 0.044, with an R merge of 0.256 in the highest resolution shell. No evidence of twinning was observed. Molecular replacement employing the unliganded AgT-DHDPS structure as a search model showed an unambiguous solution with two monomers in the asymmetric unit with a translation-function Z-score (TFZ) of 34.6 and a final log-likelihood gain (LLG) of 9156.

Further model building for each of these structures is under way, although we have yet to confirm that the ligands are present in the relevant models. Determination of these crystal structures will offer insight into the design of novel pesticide agents.

Acknowledgments

This research was undertaken on the MX1 and MX2 beamlines at the Australian Synchrotron, Victoria, Australia. We would like to acknowledge Eleonora Puglia at the Department of Microbiology and Immunology, The University of Melbourne for providing the A. tumefaciens (for genomic DNA extraction) used in this study, the support and assistance of the friendly staff at the CSIRO Collaborative Crystallization Centre at CSIRO Material Science and Engineering, Parkville, Melbourne and the MX beamline scientists at the Australian Synchrotron. We would also like to thank all members of the Perugini laboratory for helpful discussions during the preparation of this manuscript. Finally, we acknowledge the Australian Research Council for providing a Future Fellowship for MAP and The University of Melbourne for providing project funding (FRGSS 2011 project grant). RCJD acknowledges (i) the C. R. Roper Bequest for Fellowship support, (ii) the New Zealand Royal Society Marsden Fund for funding support, in part (contract UOC1013), and (iii) the US Army Research Laboratory and US Army Research Office under contract/grant No. W911NF-11-1-0481 for support, in part.

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