Structure of dihydrodipicolinate synthase from Methanocaldococcus jannaschii

The crystal structure of dihydrodipicolinate synthase from the (S)-lysine synthesis pathway of Methanocaldococcus jannaschii has been solved to 2.2 Å resolution, revealing a functional homotetramer.

Abstract

In bacteria and plants, dihydrodipicolinate synthase (DHDPS) plays a key role in the (S)-lysine biosynthesis pathway. DHDPS catalyzes the first step of the condensation of (S)-aspartate-β-semialdehyde and pyruvate to form an unstable compound, (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. The activity of DHDPS is allosterically regulated by (S)-lysine, a feedback inhibitor. The crystal structure of DHDPS from Methanocaldococcus jannaschii (MjDHDPS) was solved by the molecular-replacement method and was refined to 2.2 Å resolution. The structure revealed that MjDHDPS forms a functional homo­tetramer, as also observed in Escherichia coli DHDPS, Thermotoga maritima DHDPS and Bacillus anthracis DHDPS. The binding-site region of MjDHDPS is essentially similar to those found in other known DHDPS structures.

1. Introduction

In prokaryotes and higher plants, DHDPS (DapA; dihydrodipico­linate synthase; EC 4.2.1.52) catalyzes the first unique step leading to (S)-lysine biosynthesis via the diaminopimelate pathway: the con­densation of pyruvate and (S)-aspartate β-semialdehyde into (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid (HTPA; see Dobson et al., 2004 ▶, and references therein). Lysine, the final product of the lysine-biosynthesis pathway, and the precursor diaminopimelic acid (DAP) are both essential for bacterial survival and growth. Lysine is important for protein biosynthesis and DAP is a structural cross-linking component of the peptidoglycan layer of mycobacterial cell walls (Cummins & Harris, 1956 ▶). The unstable product HTPA is obtained in the first catalysis step (Blickling et al., 1997 ▶). Dihydro­dipicolinic acid can be formed spontaneously from HTPA by the removal of water.

As DapA/DHDPS is expressed in plants and microorganisms but not in animals, this enzyme is a potential target candidate for antibiotics and herbicides (Coulter et al., 1999 ▶; Cox et al., 2000 ▶; Hutton et al., 2003 ▶); however, no potent inhibitor is available to date. We now report the crystal structure of DHDPS from Methanocaldococcus jannaschii (MjDHDPS) determined at 2.2 Å resolution.

2. Methods and materials

2.1. Cloning, expression and purification of MjDHDPS

The gene encoding MjDHDPS (gi:15668419) was amplified via PCR using M. jannaschii DSM 2661 genomic DNA and was cloned into the pET-21a expression vector (Merck Novagen). The expression vector was introduced into Escherichia coli Rosetta (DE3) strain (Merck Novagen) and the recombinant strain was cultured in LB medium containing 30 µg ml⁻¹ chloramphenicol and 50 µg ml⁻¹ ampicillin. The cells (16.4 g) from 4.5 l of medium were collected by centrifugation and were lysed by sonication in 32 ml 20 mM Tris–HCl buffer pH 8.0 containing 500 mM NaCl, 5 mM β-mercaptoethanol and 1 mM phenylmethylsulfonyl fluoride on ice. The cell lysate was heat-treated at 363 K for 13 min and centrifuged at 15 000g for 30 min at 277 K. The supernatant was desalted by fractionation on a HiPrep 26/10 column (GE Healthcare Biosciences). The sample was applied onto a Toyopearl SuperQ-650M column (Tosoh, Tokyo) equilibrated with 20 mM Tris–HCl buffer pH 8.0 and was eluted with a linear (0–0.4 M) gradient of NaCl. The target sample, which eluted in the 0.28 M NaCl fraction, was then applied onto a Resource Q column (GE Healthcare Biosciences) equilibrated with 20 mM Tris–HCl buffer pH 8.0 and was eluted with a linear gradient of 0–0.4 M NaCl. The fractions that eluted in 0.29 M NaCl were further purified using a hydroxyapatite CHT5-I column (Bio-Rad Laboratories) with a linear gradient of 0.01–0.5 M potassium phosphate buffer pH 7.0. The target sample, which eluted in the 0.11 M potassium phosphate fraction, was concentrated and applied onto a HiLoad 16/60 Superdex 200 pg column (GE Healthcare Biosciences) equilibrated with 20 mM Tris–HCl buffer pH 8.0 containing 200 mM NaCl. The protein sample was analyzed by SDS–PAGE and was confirmed by N-­terminal amino-acid sequencing. After concentration to 44.2 mg ml⁻¹ by ultrafiltration, the yield was 8.8 mg from 16.4 g of cells.

2.2. Crystallization

Crystallization was performed by the oil-batch method at 291 K. A 0.5 µl aliquot of crystallization reagent, consisting of 0.1 M cacodylate buffer pH 6.5 containing 18%(w/v) PEG 8000 and 0.2 M calcium acetate (Crystal Screen 1 condition No. 44; Hampton Research), was mixed with 0.5 µl 44.2 mg ml⁻¹ protein solution. The mixture was then covered with 15 µl of silicone and paraffin oil. Crystals suitable for X-ray data collection appeared within 3 d and reached final dimensions of 0.1 × 0.08 × 0.08 mm (Fig. 1 ▶ a).

Figure 1.

Overall tertiary and quaternary structures of MjDHDPS. (a) Crystals of the MjDHDPS protein. (b) The quaternary structure of the DHDPS tetramer coloured by subunit. (c) Stereoview of a ribbon diagram of the DHDPS subunit, comprising the (β/α)₈-barrel and the C-terminal domain, and coloured from blue (N-terminus) to red (C-­terminus). The view is looking down the axis of the (β/α)₈-barrel. All figures were produced using PyMOL (DeLano, 2002 ▶) unless mentioned otherwise.

2.3. Data collection and processing

Diffraction data were collected using a MAR Mosaic 225 mm CCD detector on the PX10.1 beamline at the Daresbury Synchrotron Radiation Source (SRS), England. The crystals were directly flash-cooled in a nitrogen-gas stream at 100 K using 10%(v/v) ethylene glycol as a cryoprotectant in a drop made up of equal volumes of the protein and reservoir solutions. The crystals were maintained at 100 K during data collection. The crystal-to-detector distance was set to 150 mm. The diffraction data were processed with the HKL-2000 software suite (DENZO and SCALEPACK; Otwinowski & Minor, 1997 ▶). Crystallographic data and refinement statistics are summarized in Table 1 ▶.

Table 1. Summary of data-collection and refinement statistics.

Values in parentheses are for the highest resolution shell.

Data collection
Source	SRS, PX10.1
Wavelength (Å)	0.979
Space group	P21
Unit-cell parameters (Å, °)	a = 80.5, b = 76.5, c = 101.9, γ = 106.9
Resolution (Å)	50–2.20 (2.28–2.20)
Completeness (%)	97.6 (82.6)
Redundancy	3.9 (2.7)
No. of independent observations	58517 (4918)
Rmerge† (%)	6.7 (21.1)
Refinement statistics
Resolution limit (Å)	20–2.2
R factor/Rfree‡ (%)	15.8/22.4
Mean B factor (Å2)	26.9
Wilson B factor (Å2)	27.9
No. of refined atoms	8852
No. of water molecules	700
R.m.s. deviations
Bond lengths (Å)	0.022
Bond angles (°)	1.8

^† R _merge = .

^‡ R = . R _free was calculated with 5% of data that were omitted from refinement.

2.4. Structure determination and refinement

The structure of MjDHDPS was solved by the molecular-replacement method with the program MOLREP (Vagin & Teplyakov, 1997 ▶), incorporated in the CCP4 suite (Collaborative Computational Project, Number 4, 1994 ▶), using the T. maritima DHDPS structure (PDB code 1o5k) as a search model. The structure solution confirmed that there were four subunits with approximate 222 symmetry in the asymmetric unit (Fig. 1 ▶ b). The model was refined with CNS (Brünger et al., 1998 ▶) and warpNtrace refinement in ARP/wARP was subsequently performed (Morris et al., 2003 ▶). Surprisingly, tracing using the warpNtrace method yielded almost complete chains of the MjDHDPS quaternary structure. Several further rounds of manual fitting and refitting were performed using the program Coot (Emsley & Cowtan, 2004 ▶), in combination with refinement using REFMAC5 (Murshudov et al., 1997 ▶), with careful inspection of the 2F _o − F _c, F _o − F _c and OMIT electron-density maps. The refined model of the homotetrameric form consists of 1853 residues and 700 water molecules, with a final R _work of 15.8% and an R _free of 22.4% at 2.2 Å resolution. The stereochemistry of the complex structure is excellent, as assessed by PROCHECK (Laskowski et al., 1993 ▶). Ramachandran plot analysis of this structure revealed that 92.6% of the residues are in the allowed region and 7.0% are in the generously allowed region, with 0.4% in the disallowed region. The functional residue Tyr106 lies in the disallowed region, as was also observed for the equivalent position in other DHDPS structures. The refinement statistics are summarized in Table 1 ▶.

3. Results and discussion

3.1. Overall structure

The asymmetric unit contains four MjDHDPS molecules (Fig. 1 ▶ c). The overall tertiary structure of MjDHDPS possesses a (β/α)₈-barrel fold (or TIM barrel, named after the structure of triosephosphate isomerase) with three additional α-helices (α9–α11) at the C-­terminus of the chain (Figs. 1 ▶ c and 2 ▶). As in other TIM-barrel family proteins, the β-strands of the barrel form an intrinsic network of hydrogen-bonding interactions with the neighbouring β-­strands and are oriented in the same directions. The overall twist adopted by all of the strands pushes the first and eighth strands close together, which is subsequently responsible for the closure of the barrel by the formation of hydrogen bonds between the main-chain atoms of the β1 and β8 β-strands.

Figure 2.

Sequence alignment of DHDPS proteins from T. maritima (DAPA_THEMA), E. coli (DAPA_ECOLI) and B. anthracis (Q81WN7_BAC) with MjDHDPS (DAPA_METJA). The secondary-structural features of MjDHDPS are indicated above the alignment. The potentially important residues in the active-site region and in the allosteric lysine-binding site region are indicated by blue circles and red triangles, respectively. The colours reflect the similarity (white characters on a red background represent conserved residues, red characters represent similarity in a group and blue frames indicate similarity across groups). The sequence was aligned and displayed using ClustalW (Thompson et al., 1994 ▶) and ESPript (Gouet et al., 1999 ▶), respectively.

The asymmetric unit comprises four subunits. As observed in E. coli DHDPS (Mirwaldt et al., 1995 ▶), T. maritima DHDPS (Pearce et al., 2006 ▶) and B. anthracis DHDPS (Blagova et al., 2006 ▶), the four subunits of the asymmetric unit clearly form a homotetramer with approximate 222 symmetry (Fig. 1 ▶ b). The homotetramer, which is composed of a dimer of ‘tight dimers’, is functionally important for its catalytic activity. The A–B dimer interface is formed by the segments β3–α3, β4–α4, β5–α5, α9–α10 and α10–α11 from each monomer, which contribute a buried surface area of about 2776 Ų. The A–D dimer association mainly arises from interhelical interactions. The dimer interface is primarily composed of the α6, α7 and α9 helices and the segments α7–β8 and α8–α9 from each monomer. The α6 and α7 helices of chain A interact with α7 of chain D, while α6 of chain D interacts with α7 and α9 of chain A. These interfaces are less extensive, with an average buried surface of 1918 Ų. There are no contacts between subunits A and C or between subunits B and D, and the tetramer contains a large central cavity (Fig. 1 ▶ b). As observed in the other bacterial enzymes, the active site is located in the centre of the (β/α)₈-barrel in each monomer. The functional residue Lys161, which participates in Schiff-base formation, is located within the β-­barrel and the side chain of Tyr132 sits over this residue. The allosteric lysine-binding site is located in the cleft at the tight-dimer interface, with each lysine molecule coordinated by residues from each monomer within the tight dimer.

3.2. Comparison with other DHDPS structures

As expected, a DALI (Holm & Sander, 1993 ▶) search of the MjDHDPS structure against the structures in the Protein Data Bank revealed that the MjDHDPS structure is essentially similar to other known DHDPS structures. The highest Z-score value of 45.9 was obtained for T. maritima DHDPS (PDB code 1o5k; Pearce et al., 2006 ▶), followed by 44.9 and 43.3 for B. anthracis DHDPS (PDB code 1xky; Blagova et al., 2006 ▶) and E. coli DHDPS (PDB code 1dhp; Mirwaldt et al., 1995 ▶), respectively. Superposition of the MjDHDPS structure onto the structures of T. maritima DHDPS, B. anthracis DHDPS and E. coli DHDPS yielded r.m.s.d. values of 1.1, 1.2 and 1.4 Å, respectively, for all C^α atoms (Fig. 3 ▶ a).

Figure 3.

Comparison with other DHDPS structures. (a) Superposition of MjDHDPS (raspberry) onto the E. coli DHDPS (pink), B. anthracis DHDPS (yellow) and T. maritima DHDPS (cyan) structures for the C^α atoms corresponding to the (β/α)₈-barrel and the C-terminal domain. (b) Superposition of the active sites of the MjDHDPS (wheat) and T. maritima DHDPS (cyan) structures. The numbering is shown for the M. jannaschii enzyme. (c) Superposition of the (S)-lysine-binding sites of E. coli DHDPS (blue) and MjDHDPS (wheat). The (S)-lysine molecule is shown in green. The numbering is shown for the M. jannaschii enzyme.

3.3. The catalytic site region

The mechanism of the E. coli DHDPS reaction has been studied in detail (Blickling et al., 1997 ▶). Immediately after the enzyme binds to the pyruvate substrate, a Schiff base is formed between the substrate and the active-site lysine residue Lys161. The second substrate, aspartate semialdehyde, then binds. This is followed by dehydration and cyclization to form the product, which is subsequently released. It has been proposed that Tyr133 (Tyr132 in MjDHDPS), Thr44 and Thr107 (Tyr106 in MjDHDPS) form a catalytic triad that shuttles protons to and from the active site (Dobson et al., 2004 ▶; Fig. 3 ▶ b).

(S)-Lysine is an allosteric modulator of DHDPS function and partially inhibits the catalytic activity. Dobson et al. (2005 ▶) recently determined the high-resolution crystal structure of the E. coli DHDPS–(S)-lysine complex, which revealed that the (S)-lysine-binding site is located in a crevice at the interface of the tight dimer, which is distal from the active site but connected to the active site by a conserved water channel. The allosteric modulator (S)-lysine is surrounded by Ala49, His53, His56, Gly78, Asn80, Glu84 and Tyr106 in E. coli DHDPS (Figs. 2 ▶ and 3 ▶ c).

A comparison of the catalytic site region of MjDHDPS with those of E. coli DHDPS, T. maritima DHDPS and B. anthracis DHDPS revealed that the regions are very similar (Fig. 3 ▶ b). The potential interacting residues and the catalytic site region are highly conserved in the MjDHDPS structure, suggesting that the catalytic function of MjDHDPS may be similar to those of E. coli DHDPS, T. maritima DHDPS and B. anthracis DHDPS. However, further biochemical and structural analyses of MjDHDPS complexes are required in order to understand the enzymatic mechanism of this protein.

Supplementary Material

PDB reference: dihydrodipicolinate synthase, 2yxg, r2yxgsf