Cloning, expression, purification, crystallization and X-ray diffraction analysis of dihydrodipicolinate synthase from the human pathogenic bacterium Bartonella henselae strain Houston-1 at 2.1 Å resolution

The enzyme dihydrodipicolinate synthase catalyzes the committed step in the synthetic pathway for- the intermediate diaminopimelate and the essential amino acid lysine in bacteria and plants. Here, the crystallization and X-ray diffraction analysis of dihydrodipicolinate synthase from the human pathogenic bacterium B. henselae, the causative bacterium of cat-scratch disease, are presented.

Abstract

The enzyme dihydrodipicolinate synthase catalyzes the committed step in the synthesis of diaminopimelate and lysine to facilitate peptidoglycan and protein synthesis. Dihydrodipicolinate synthase catalyzes the condensation of l-aspartate 4-semialdehyde and pyruvate to synthesize l-2,3-dihydrodipico­linate. Here, the cloning, expression, purification, crystallization and X-ray diffraction analysis of dihydrodipicolinate synthase from the pathogenic bacterium Bartonella henselae, the causative bacterium of cat-scratch disease, are presented. Protein crystals were grown in conditions consisting of 20%(w/v) PEG 4000, 100 mM sodium citrate tribasic pH 5.5 and were shown to diffract to ∼2.10 Å resolution. They belonged to space group P2₁2₁2₁, with unit-cell parameters a = 79.96, b = 106.33, c = 136.25 Å. The final R values were R _r.i.m. = 0.098, R _work = 0.183, R _free = 0.233.

1. Introduction  

Enzymes involved in the bacterial synthesis of diamino­pimelate/lysine (DAP/Lys) are attractive targets for the development of bactericidal compounds, especially from bacteria that are deemed to be pathogenic. The Gram-negative bacterium Bartonella henselae is the causative agent of cat-scatch disease (CSD; Mazur-Melewska et al., 2015 ▸). There are 25 species in the genus and 50% of the species have been implicated in CSD (Breitschwerdt & Kordick, 2000 ▸). The reservoirs of B. henselae include, but are not limited to, cats, guinea pigs and rabbits; the majority of CSD infections are transmitted by cats based on a study in Poland that showed positive IgG antibodies for the bacterium in 50–90% of cats tested (Podsiadły et al., 2002 ▸). The symptoms of CSD can include skin inflammation, fever, lymphadenopathy, various eye disorders, encephalitis and endocarditis (Mazur-Melewska et al., 2015 ▸).

There are four variants of the diaminopimelate DAP/Lys pathway in bacteria. Using l-aspartate (Asp) as the starting molecule, all four variants share the common enzymatic steps for the synthesis of tetrahydrodipicolinate (THDP) from Asp (Hudson et al., 2006 ▸). The synthesis of the penultimate compound meso-diaminopimelate (m-DAP) from THDP defines the uniqueness of the variants. In most bacteria, acetylated or succinylated (acyl) intermediates are used in the conversion of THDP to l,l-diaminopimelate (l,l-DAP) employing the enzymes DapD, DapC and DapE (Fig. 1 ▸). In another variant, a few bacterial lineages employ the enzyme meso-diaminopimelate (m-DAP) dehydrogenase (Ddh), which catalyzes the synthesis of m-DAP from THDP in a single reaction, circumventing the acylase, aminotransferase, deacylase and epimerase enzymatic steps used in the acyl pathway (Fig. 1 ▸). A novel pathway was recently discovered in plants and subsequently in certain bacterial lineages that employs the enzyme l,l-DAP aminotransferase (DapL). DapL utilizes the acid glutamate as the amino donor and THDP as the amino acceptor to synthesize l,l-DAP in a single step circumventing the acylase, aminotransferase and deacyl­ase reactions that are present in the acyl pathway (Hudson et al., 2006 ▸, 2008 ▸; McCoy et al., 2006 ▸; Nachar et al., 2012 ▸). The conversion of m-DAP to lysine by the enzyme m-DAP decarboxylase (LysA) is the final step in the pathway and is common to all four variants (Fig. 1 ▸).

Figure 1.

Variants of the diaminopimelate/lysine-synthesis pathways in bacteria. (a) The synthesis of THDP from aspartate, (b) the acyl pathway, (c) the meso-diaminopimelate dehydrogenase (Ddh) pathway and (d) the diaminopimelate aminotransferase (DapL) pathway. Abbreviations are as follows: LysC, aspartate kinase; Asd, aspartate semialdehyde dehydrogenase; DapA, dihydrodipicolinate synthase; DapB, dihydrodipicolinate reductase; DapD, 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-acyltransferase); DapC, acyl-diaminopimelate aminotransferase; DapE, acyl-diaminopimelate deacylase; Ddh, meso-diaminopimelate dehydrogenase; DapL, l,l-diaminopimelate aminotransferase; DapE, diaminopimelate epimerase; LysA, meso-diaminopimelate decarboxylase; MurE, UDP-N-acetylmuramoylalanyl-d-glutamate-2,6-diaminopimelate ligase; LysU, lysine-tRNA synthetase.

The enzyme dihydrodipicolinate synthase (DapA; EC 4.2.1.52) catalyzes the committed step in the DAP/Lys path­way in bacteria. The enzyme is responsible for the synthesis of dihydrodipicolinate via a condensation reaction with the substrates pyruvate and l-aspartate 4-semialdehyde to synthesize l-2,3-dihydrodipicolinate (Fig. 2 ▸). Based on the fact that the gene is essential in bacteria and is a putative target for antibiotic development, there have been numerous studies regarding the structure of DapA from a number of pathogenic bacteria such as Escherichia coli, Mycobacterium tuberculosis, Neisseria meningitidis, Pseudomonas aeruginosa, Staphylococcus aureus and Bacteriodes thetaiotaomicron, which is an endosymbiont of the human gut (Dobson et al., 2005 ▸; Kefala et al., 2008 ▸; Devenish et al., 2009 ▸; Kaur et al., 2011 ▸; Burgess et al., 2008 ▸; Girish et al., 2008 ▸; Mank et al., 2015 ▸). Although there has been a move to change the name of the enzyme to 4-hydroxy-tetrahydrodipicolinate synthase, we will use the more common enzyme name here (Bochaute et al., 2013 ▸).

Figure 2.

The reaction catalyzed by dihydrodipicolinate synthase (DapA) showing the condensation of l-aspartate 4-semialdehyde and pyruvate to form l-2,3-dihydrodipicolinate.

The de novo biosynthesis of DAP/Lys is absent in all animals, particularly humans, since their genomes do not contain the machinery necessary for the synthesis of DAP/Lys. As such, Lys is a deemed to be an essential amino acid and must be acquired through dietary means for protein synthesis. In addition, in bacteria, the penultimate intermediate m-DAP serves as a cross-linking amino acid in the synthesis of peptidoglycan (PG) in most Gram-negative bacteria, and Lys serves the same role in most Gram-positive bacteria (Hutton et al., 2007 ▸). Therefore, since Lys and DAP are necessary for both PG and protein synthesis, the inhibition of enzymes associated with this pathway will lead to cell death via lysis as a result of the osmotic pressure from the lack of PG (Cox, 1996 ▸; Baizman et al., 2000 ▸). The lack of protein synthesis would also lead to a bactericidal or bacteriostatic effect since Lys is one of the 20 ubiquitous proteogenic amino acids (Triassi et al., 2014 ▸). Therefore, enzymes associated with the DAP/Lys pathway are attractive targets for the development of antibiotics, herbicides and algaecides. As such, the macromolecular structure of DapA from pathogenic bacteria affords the opportunity for the identification and or development of compounds that are deemed to be bactericidal. Here, we present the cloning, expression, purification, crystallization and X-ray diffraction analysis of dihydrodipicolinate synthase from the human pathogenic bacterium B. henselae strain Houston-1 (BhDapA), the causative agent of cat-scratch disease.

2. Materials and methods  

2.1. Amplification and cloning of DapA from B. henselae  

Cloning, expression and purification were conducted as part of the Seattle Structural Genomics Center for Infectious Disease (SSGCID) following standard protocols described previously (Myler et al., 2009 ▸; Stacy et al., 2011 ▸; Serbzhinskiy et al., 2015 ▸; Bryan et al., 2011 ▸; Choi et al., 2011 ▸). The full-length open reading frame (ORF) encoding amino acids 1–294 (UniProt Q6G468) was PCR-amplified from B. henselae strain Houston-1 genomic DNA (ATCC 49882D-5). The ORF was amplified using the following primer sequences: Bh-dapA-F, 5′-GGGTCCTGGTTCGATGCTCAAGGGAGCTGTGACCG-3′, and Bh-dapA-R, 5′-CTTGTTCGTGCTGTTTATTATTCTTTAAGCAAACCCGCATGGTA-3′. The ORF was cloned into the ligation-independent cloning (LIC) expression vector pAVA0421 encoding a cleavable 6×His fusion tag followed by the human rhinovirus 3C protease-cleavage sequence (MAHHHHHHMGTLEAQTQGPGS) followed by the ORF (Aslanidis & de Jong, 1990 ▸). The human rhinovirus 3C protease-cleavage site is between the glutamine and glycine residues that are underlined. Plasmid DNA was transformed into chemically competent E. coli BL21 (DE3) R3 Rosetta cells. The plasmid containing His-Bh-dapA was tested for expression and 2 l of culture were grown using auto-induction medium in a LEX Bioreactor (Epiphyte Three Inc.) as described previously (Studier, 2005 ▸; Choi et al., 2011 ▸).

2.2. Protein production and purification  

His-tagged BhDapA was purified in a four-step protocol consisting of an Ni²⁺-affinity chromatography (IMAC) step, cleavage of the N-terminal His tag with 3C protease, removal of the cleaved His tag by passage over a second Ni²⁺-affinity chromatography column and size-exclusion chromatography (SEC). All chromatographic steps were performed on an ÄKTApurifier 10 (GE Healthcare) using automated IMAC and SEC programs according to previously described procedures (Serbzhinskiy et al., 2015 ▸; Bryan et al., 2011 ▸). Bacterial pellets were thawed into 200 ml lysis buffer consisting of 25 mM HEPES pH 7.0, 500 mM NaCl, 5% glycerol, 0.5% CHAPS, 30 mM imidazole, 10 mM MgCl₂, 1 mM TCEP, 250 µg ml⁻¹ AEBSF, 0.025% sodium azide and were lysed by sonication. Benzonase (20 µl, 25 units µl⁻¹) was added and the crude lysate was mixed at room temperature for 45 min. The lysate was then clarified by centrifugation at 10 000 rev min⁻¹ for 1 h using a Sorvall centrifuge (Thermo Scientific). The clarified supernatant was then passed over an Ni–NTA HisTrap FF 5 ml column (GE Healthcare) with a mobile phase consisting of loading buffer composed of 25 mM HEPES pH 7.0, 500 mM NaCl, 5% glycerol, 30 mM imidazole, 1 mM TCEP, 0.025% sodium azide. The column was washed with 20 column volumes (CV) of loading buffer and was eluted with loading buffer plus 250 mM imidazole in a linear gradient over 7 CV. His-MBP-3C protease was added to the eluted protein at a ratio of 1:50 to cleave the His tag. The protein was then transferred into a dialysis bag and dialyzed overnight at 4°C in 1 l of 3C dialysis buffer consisting of 25 mM HEPES pH 7.5, 200 mM NaCl, 5% glycerol, 1 mM TCEP. After overnight dialysis, the concentrations of imidazole and sodium chloride in the reaction mixture were adjusted to 30 and 500 mM, respectively, and the dialysate was passed over a second Ni²⁺-affinity column to remove His-MBP-3C protease, uncleaved protein and cleaved His tag. The cleaved protein was recovered in the flowthrough and concentrated prior to loading onto the final SEC column. A SEC column (Superdex 75, GE Healthcare) was equilibrated with running buffer composed of 20 mM HEPES pH 7, 0.3 M NaCl, 5% glycerol, 1 mM TCEP. The SEC chromatogram showed one predominant peak which was separated from a 70 kDa impurity (Fig. 3 ▸). The retention time of the primary peak was used to estimate the molecular weight by comparison with known standards (data not shown). The estimated molecular weight was consistent with a tetrameric assembly in buffer solution. The peak fractions were collected and analyzed for the presence of the protein of interest using SDS–PAGE in a 4–20% gradient gel with Tris–MOPS running buffer for 40 min at 175 V. The peak fractions were pooled and concentrated to 25.2 mg ml⁻¹ using an Amicon (Millipore) purification system (Fig. 3 ▸). Aliquots of 100 µl were cryocooled in liquid nitrogen and stored at −80°C until use for crystallization.

Figure 3.

SDS–PAGE analysis of purified recombinant DapA. (a) Size-exclusion chromatography (SEC) chromatogram of BhDapA from the final purification column showing a large peak with a small shoulder. (b) SDS–PAGE gel of peak fractions. Fractions 21–26 were pooled, concentrated to 25.2 mg ml⁻¹ and used for crystallization trials. The proteins fractions were resolved on a gradient SDS–PAGE gel (4–20%) and stained using Coomassie Blue for visualization.

2.3. Crystallization and data collection  

Purified BhDapA was screened for crystallization in 96-well sitting-drop plates against the Wizard Classic 1 & 2 and Wizard Classic 3 & 4 crystal screens (Rigaku Reagents). Equal volumes of protein solution (0.4 µl) and precipitant solution were set up at 293 K against reservoir (80 µl) in sitting-drop vapor-diffusion format. The final crystallization precipitant was Wizard Classic 3 & 4 condition B2 composed of 20% PEG 4000, 100 mM sodium citrate tribasic pH 5.5. The crystals were cryoprotected in crystallant plus 25% 1,2-ethanediol and were cryocooled by dipping them into liquid nitrogen. Data were collected under the Collaborative Crystallography program of the Berkeley Center for Structural Biology at the Advanced Light Source, Berkeley National Laboratory. Data were collected at 100°C on Advanced Light Source beamline 5.0.1 using an ADSC Quantum 315 CCD detector with 1° oscillations at a wavelength of 0.9774 Å. Data were reduced with HKL-2000 (Otwinowski & Minor, 1997 ▸). Raw X-ray diffraction images are available at the Integrated Resource for Reproducibility in Macromolecular Crystallography at http://www.proteindiffraction.org.

2.4. Structure solution and refinement  

The structure was solved by molecular replacement with Phaser (McCoy et al., 2007 ▸) from the CCP4 suite of programs (Winn et al., 2011 ▸) using PDB entry 2rfg (B. S. Kang, M. H. Kim, G. H. Kim & K. J. Kim, unpublished work) as a search model. The structure was refined using iterative cycles of REFMAC5 (Murshudov et al., 2011 ▸) followed by manual rebuilding of the structure using Coot (Emsley et al., 2010 ▸). The quality of all of the structures was checked using MolProbity (Chen et al., 2010 ▸). Data-collection and data-reduction and refinement statistics are shown in Tables 1 ▸ and 2 ▸, respectively. The structure was refined to a resolution of 2.1 Å. Unambiguous electron density was visible for the placement of 1,2-ethanediol, which was used as a cryoprotectant during cryocooling of the crystals (Fig. 4 ▸). Structure figures were analyzed and prepared using PyMOL (v.1.5; Schrödinger) and PISA (Krissinel & Henrick, 2007 ▸). PISA analysis predicts a stable tetrameric assembly composed of four monomers, A, B, C and D, with a buried surface of 10 384 Ų. Stable dimers are predicted to be formed by molecules A and C and molecules B and D. The average buried surface of the AC and BD dimers was 3420 Ų (Fig. 5 ▸; dimer interface), whereas the average buried surface of the AB and CD dimers was 2335 Ų (Fig. 5 ▸; tetramer interface). Coordinates and structure factors have been deposited with the Protein Data Bank (PDB; http://www.rcsb.org) as entry 3si9.

Table 1. Data collection and processing.

Values in parentheses are for the outer shell.

Diffraction source	Beamline 5.0.1, ALS
Wavelength (Å)	0.9774
Temperature (K)	100.0
Detector	ADSC Quantum 315r CCD
Crystal-to-detector distance (mm)	250
Rotation range per image (°)	1
Total rotation range (°)	150
Exposure time per image (s)	10
Space group	P212121
a, b, c (Å)	79.96, 106.33, 136.25
Mosaicity (°)	0.86
Resolution range (Å)	50.00–2.10 (2.14–2.10)
Total No. of reflections	417245 (20757)
No. of unique reflections	68289 (3414)
Completeness (%)	99.8 (100.0)
Multiplicity	6.1 (6.1)
〈I/σ(I)〉	18.3 (3.5)
R r.i.m. †	0.098 (0.457)
Overall B factor from Wilson plot (Å2)	29.0

^†Estimated R _r.i.m. = R _merge[N/(N − 1)]^1/2, where N is the data multiplicity.

Table 2. Structure solution and refinement.

Values in parentheses are for the outer shell.

Resolution range (Å)	50.00–2.10 (2.15–2.10)
Completeness (%)	99.4 (97.2)
σ Cutoff	F > 0.000σ(F)
No. of reflections, working set	67983 (4586)
No. of reflections, test set	3445 (247)
Final R cryst	0.183 (0.227)
Final R free	0.233 (0.300)
No. of non-H atoms
Protein	8672
Ligand	36
Water	560
Total	9268
R.m.s. deviations
Bonds (Å)	0.014
Angles (°)	1.440
Average B factors (Å2)
Protein	28.6
Ligand	39.7
Water	33.9
Ramachandran plot
Most favored (%)	97.7
Outliers (%)	0

Figure 4.

Electron density showing the placement of the 1,2-ethanediol solvent molecule. The molecule is highlighted by the yellow arrow.

Figure 5.

Three-dimensional view of BhDapA (PDB entry 3si9) showing the tetrameric structure. The individual monomeric units are shown by the color scheme, with monomer A in red, monomer B in yellow, monomer C in green and monomer D in blue. The yellow arrow shows the ligand 1,2-ethanediol. The dimeric and tetrameric assembly interfaces are highlighted with black arrows.

3. Results and discussion  

The DapA ortholog from B. henselae strain Houston-1 was successfully cloned, expressed and purified to homogeneity using a four-step purification protocol to a final concentration of 25.2 mg ml⁻¹. The purity of the enzyme was assessed by size-exclusion chromatography (SEC) and SDS–PAGE analysis (Fig. 3 ▸) and was suitable for crystallization trials.

Purified BhDapA was screened for crystallization in 96-well sitting-drop plates. The structure was subsequently refined to a resolution of 2.1 Å. The coordinates have been deposited in the PDB as entry 3si9. The three-dimensional structure of BhDapA is tetrameric in nature, consisting of four monomeric units that interact via a putative ‘dimer interface’ and a ‘tetramer assembly’ interface (Fig. 5 ▸). This is consistent with the structures of DapA orthologs from the bacteria E. coli (Blickling & Knäblein, 1997 ▸; Dobson et al., 2005 ▸; Mirwaldt et al., 1995 ▸), Bacillus anthracis (Blagova et al., 2006 ▸) and Thermotoga maritima (Pearce et al., 2006 ▸) in addition to the eukaryotic orthologs from the plant species Nicotiana sylvestris (Blickling et al., 1997 ▸) and Vitis vinifera (Atkinson et al., 2012 ▸). Like the E. coli ortholog, each monomer of BhDapA consists of two domains in which each polypeptide possesses a (β/α)₈-barrel structure connected to a predominantly α-helical C-terminal domain (Mirwaldt et al., 1995 ▸; Fig. 6 ▸). The arrangement of the monomeric units forms a large cavity in the center where the two dyads connect (Fig. 6 ▸). The structural features of the large cavity are conserved in bacterial orthologs but not in plant orthologs, where the dyads interact in a ‘back-to-back’ orientation that prevents cavity formation, as shown in the structures from N. sylvestris and V. vinifera (Atkinson et al., 2012 ▸). A common feature of the bacterial enzyme is the presence of a conserved lysine residue in the active site. This lysine residue is involved in the catalytic mechanism of the enzyme through the formation of an enamine linkage via a Schiff base to the substrate (pyruvate) and the catalytic triad (Dobson et al., 2004 ▸). The conserved lysine residue at position 166 in the structure (PDB entry 3si9) is shown in Fig. 6 ▸. The structure of the DapA ortholog from Agrobacterium tumefaciens (AtDapA) has been solved (PDB entries 4i7w, 4i7v and 4i7u; S. C. Atkinson, C. Dogovski, R. C. J. Dobson & M. A. Perugini, unpublished work) with a pyruvate covalently linked to the conserved lysine residue involved in the Schiff-base reaction to facilitate substrate catalysis. As such, the active site is clearly defined in the A. tumefaciens structures. An overlay of BhDapA with the A. tumefaciens ortholog clearly shows the conserved lysine residue at position 166, highlighting the active site of BhDapA (Fig. 7 ▸).

Figure 6.

Graphical representation of the secondary features of BhDapA (PDB entry 3si9). (a) depicts the various α-helices and β-barrels that comprise one of the four polypeptides. The black arrow highlights the methionine residue that is indicative of the translation start site and the black circle highlights the conserved lysine residue in the active site of the enzyme that is involved in catalysis at position 166. (b) Graphical key depicting the secondary-structural features of the enzyme.

Figure 7.

Comparative overlay of BhDapA (PDB entry 3si9; green) on AtDapA (PDB entry 4i7w; blue) bound to pyruvate (black arrow) depicting the active site of BhDapA by highlighting the conserved lysine residue at position 166 (yellow arrow) involved in the Schiff-base reaction involved in substrate catalysis.

In summary, we present the cloning, purification, crystallization and X-ray diffraction analysis of DapA from the pathogenic bacterium B. henselae strain Houston-1. The structural information gathered from the characterization of DapA from this bacterium may be used to identify specific differences between bacterial orthologs of this diaminopimelate- and lysine-biosynthetic enzyme. This information has the potential to be useful in the design or identification of compounds that possess bactericidal activity.

Supplementary Material

PDB reference: dihydrodipicolinate synthase, 3si9