Expression, purification, crystallization and preliminary X-ray analysis of two arginine-biosynthetic enzymes from Mycobacterium tuberculosis

Two enzymes responsible for arginine biosynthesis in M. tuberculosis were expressed in Escherichia coli, then purified to homogeneity. Preliminary X-ray analysis of diffraction-quality crystals grown from each enzyme are reported.

Abstract

The gene products of two open reading frames from Mycobacterium tuberculosis (Mtb) have been crystallized using the sitting-drop vapour-diffusion method. Rv1652 encodes a putative N-acetyl-γ-glutamyl-phosphate reductase (MtbAGPR), while the Rv1656 gene product is annotated as ornithine carbamoyltransferase (MtbOTC). Both MtbAGPR and MtbOTC were expressed in Escherichia coli, purified to homogeneity and crystallized. Native data for each crystal were collected to resolutions of 2.15 and 2.80 Å, respectively. Preliminary X-ray data are presented for both enzymes.

1. Introduction

Tuberculosis (TB) is a serious chronic respiratory disease caused by the pathogen Mycobacterium tuberculosis (Mtb). It is estimated that eight million people worldwide are added annually to the 2.2 billion Mtb-infected individuals (Raviglione, 2003 ▶). Despite the existence of a somewhat effective treatment administered globally by the World Health Organization (http://www.who.int/mediacentre/factsheets/fs104/en/), non-compliance with the recommended regimens has given rise to strains resistant to existing antimycobacterial chemotherapies (Espinal, 2003 ▶). A synergistic relationship also exists between HIV and TB epidemics: HIV-positive individuals are many times more likely to contract TB and TB has become the leading cause of death among AIDS sufferers (Corbett et al., 2003 ▶; Coker, 2004 ▶). The result is that approximately 6000 people die everyday from tuberculosis. The M. tuberculosis structural genomics consortium was formed in 2000 (http://www.doe-mbi.ucla.edu/TB). The goal of this consortium is to provide a structural basis for the development of new effective therapeutics for tuberculosis (Goulding et al., 2002 ▶; Smith & Sacchettini, 2003 ▶; Terwilliger et al., 2003 ▶).

In this study, we report the cloning, expression, purification and crystallization of two enzymes involved in mycobacterial arginine biosynthesis. The Rv1652 gene encodes N-acetyl-γ-glutamyl phosphate reductase (EC 1.2.1.38; MtbAGPR). AGPR catalyzes the third step of eight in the arginine-biosynthetic pathway (Cybis & Davis, 1975 ▶) by reversibly converting N-acetyl-γ-glutamyl phosphate to N-­acetyl-γ-glutamate semialdehyde (NAGSA) via an NADPH-dependent reductive dephosphorylation. Acting as a NAGSA dehydrogenase (MtbNAGSD), this enzyme catalyzes the NADP⁺-dependent oxidative phosphorylation of NAGSA (Miura-Ohnuma et al., 2005 ▶). There are four reported structures of AGPRs in the Protein Data Bank (PDB; Berman et al., 2000 ▶). The amino-acid sequences of each were compared with MtbAGPR using ClustalW (Thompson et al., 1994 ▶). The two that show the most similarity to our enzyme based on sequence are 1vkn (41% identity) from Thermotoga maritima (Joint Center for Structural Genomics, unpublished work) and 2cvo (38% identity) from Oryza sativa (Nonaka et al., 2005 ▶).

The Rv1656 gene product is annotated as ornithine carbamoyltransferase (MtbOTC; EC 2.1.3.3). This enzyme mediates the sixth step in arginine biosynthesis, specifically the carbamoylation of l-­ornithine to produce l-citrulline (Cybis & Davis, 1975 ▶; Allewell et al., 1999 ▶). Ten OTC structures from five organisms have been deposited in the PDB. The two that are most similar to MtbOTC are 1akm (39% identity) from Escherichia coli (Jin et al., 1997 ▶) and ovine OTC (1fb5; De Gregorio et al., 2003 ▶), which has 38% sequence identity.

2. Experimental methods

2.1. Cloning, expression and purification

Primers were designed using sequences for directional cloning of inserts into the Gateway cloning system (Invitrogen). They included homologous recombination sites and a region encoding a tobacco etch virus (TEV) protease-cleavage site as published elsewhere (Biswal et al., 2006 ▶). The gene-specific sequences used for these two pairs of primers are Rv1652F (5′-ATGCAAAATCGGCAGGTGGCCAATGCG-3′), Rv1652R (5′-­TCACGGCGCCACCCCCACAACCGAAAGGCC-3′), Rv1656F (5′-­GTGATCAGGCATTTCCTGCGCGACGACG-3′) and Rv1656R (5′-TCATGAGCGCTCCAGCAGCCACACCAGCAGC-3′). Mtb strain H37Rv genomic DNA in an ordered bacterial artificial chromosome library from L’Institute Pasteur (Brosch et al., 1998 ▶) was used as template to amplify the open reading frames (ORFs) by PCR. Both ORFs were inserted into the cloning vector pDONR-201 and Rv1652 was then transferred into an expression vector encoding an amino-terminal glutathione-S-transferase (GST) fusion (pDEST-15) to create the expression vector pGST-1652. Rv1656 was inserted downstream from a region encoding an N-terminal hexahistidine affinity tag in the expression plasmid pDEST-17, yielding pHIS-1656. Both expression plasmids were confirmed by DNA-sequence analysis (DNA core facility, Department of Biochemistry, University of Alberta, Canada).

GST-AGPR was expressed in E. coli BL21 (DE3) pLysS cells (Novagen). Transformed cells were incubated at 310 K until the OD_600nm = 0.8; the medium then was shifted to 295 K and expression was induced by adding isopropyl thiogalactopyranoside (IPTG) to a final concentration of 1 mM. After 18 h incubation, the cells were harvested by centrifugation at 2150g for 20 min. The bacterial pellet was resuspended in phosphate-buffered saline (PBS) with complete protease inhibitor (Roche), 2 mM DTT and 250 µg ml⁻¹ lysozyme pH 8.0. Cells were lysed by freeze–thaw and then subjected to ultrasonication. The lysate was cleared by centrifugation (30 min, 20 000g) and the supernatant was loaded onto a 5 ml GSTrap cartridge (GE Healthcare). His₆-Rv1656 was expressed in the same manner; however, harvested cells were resuspended in 20 mM Na₂HPO₄, 30 mM imidazole, 500 mM NaCl, 1 mM DTT and 250 µg ml⁻¹ lysozyme pH 7.4. Following lysis and clarification, the supernatant was loaded onto an Ni²⁺-charged 5 ml IMAC column (GE Healthcare). GST-MtbAGPR and His₆-MtbOTC were competitively eluted with either a step gradient with 10 mM glutathione or a linear gradient of imidazole from 30 to 500 mM, respectively. The GST tag was cleaved using recombinant TEV protease (Invitrogen), resulting in a full-length enzyme (352 residues) preceded by a single glycine from the expression construct. The cleaved fusion protein was loaded onto a second GST cartridge and the flowthrough was collected. MtbOTC was not proteolytically cleaved from its affinity tag; therefore, it retained 29 amino acids (MSYYHHHHHHLESTSLYKKAGSENLYFQG) from the fusion construct at the N-terminus of the full-length (307 residues) native enzyme. MtbAGPR and His₆-MtbOTC were dialyzed to change the respective buffer conditions to 20 mM Tris–HCl and 500 mM NaCl pH 7.4 for size-exclusion chromatography (SEC). The proteins were separately loaded onto a Superdex 16/60 75pg SEC column (GE Healthcare) and each protein’s purity was determined by 12% SDS–PAGE analysis (Fig. 1 ▶). MtbAGPR and MtbOTC were further confirmed by MALDI–TOF MS peptide identification following in-gel proteolysis of the excised bands. Fractions from each SEC run containing purified MtbAGPR or MtbOTC were dialyzed into 5 mM Tris–HCl pH 7.4, 50 mM NaCl and then concentrated using an Amicon Ultra 10 kDa cutoff concentrator (Millipore). The final protein concentrations were 7.5 mg ml⁻¹ and 12.5 mg ml⁻¹ for MtbAGPR and MtbOTC, respectively, as determined using a Bradford protein assay kit (Biorad) with BSA (Pierce) as a standard.

Figure 1.

12% SDS–PAGE analysis revealing the expected molecular weights for both MtbAGPR (36 kDa) and MtbOTC (33 kDa). Lane 1, molecular-weight ladder (values shown are in kDa); lane 2, purified His₆-MtbOTC; lane 3, purified MtbAGPR.

2.2. Crystallization

Initial crystallization conditions for MtbAGPR and MtbOTC were found using a Hydra Plus 1 robot (Matrix Technology) to set up trials with the sitting-drop vapour-diffusion method in 96-well format (Intelliplates, Hampton Research). Sparse-matrix screening (Jancarik & Kim, 1988 ▶) was performed using Crystal Screens I and II and the Index Screen (Hampton Research). 0.5 µl of each concentrated protein was mixed with an equal volume of reservoir solution and then incubated over 100 µl reservoirs at 273 K for 2–3 d. Optimization of crystallization conditions for MtbAGPR gave a final condition of 0.1 M Bis-Tris, 20% PEG MME 5000 pH 6.5. Diffraction-quality MtbOTC crystals were grown in 0.1 M MES, 1.6 M magnesium sulfate pH 6.5.

2.3. Data collection and processing

Crystals for X-ray data collection were first rinsed in cryoprotectant (25% glycerol in mother liquor) and then flash-frozen in liquid nitrogen. Native data sets for MtbAGPR and MtbOTC crystals were collected on beamline 8.3.1 at the Advanced Light Source at Lawrence Berkeley National Laboratory. Raw data from both were reduced, merged and scaled using the HKL-2000 program suite (Otwinowski & Minor, 1997 ▶). The crystallographic statistics of the data from the native crystals are summarized in Table 1 ▶.

Table 1. Crystal parameters and data-collection statistics for native data sets for MtbAGPR and MtbOTC.

Values in parentheses refer to the highest resolution shell.

Crystal	MtbAGPR (native)	MtbOTC (native)
Space group	P212121	P41212
Unit-cell parameters
a (Å)	89.70	136.14
b (Å)	104.03	136.14
c (Å)	138.44	141.66
α = β = γ (°)	90.0	90.0
Z†	16	24
Mosaicity (°)	0.80	0.32
Data collection
Temperature (K)	100	100
Detector	ADSC Q210	ADSC Q210
Wavelength (Å)	1.11587	0.97946
Resolution (Å)	2.15 (2.23–2.15)	2.80 (2.90–2.80)
Unique reflections	69868 (6426)	32903 (3231)
Multiplicity	3.5 (2.3)	7.7 (7.8)
I/σ(I)	15.5 (1.7)	17.8 (3.3)
Completeness (%)	98.9 (92.3)	99.2 (99.8)
Rsym (%)‡	7.3 (46.0)	10.6 (71.6)

^†No. of molecules per unit cell.

^‡ R _sym = , where I _hi is the ith observation of refection h and 〈I _h〉 is the weighted average intensity for all observations i of reflection h.

3. Results and discussion

The purifications of both MtbAGPR and MtbOTC were analyzed by 12% SDS–PAGE (Fig. 1 ▶), revealing bands of the expected molecular weight for both MtbAGPR and MtbOTC (36.3 and 33.0 kDa, respectively). Gel slices were submitted for in-gel proteolysis followed by MALDI–TOF MS identification of the resulting peptides and these confirmed the presence of the Rv1652 and Rv1656 gene products as expected.

The orthorhombic MtbAGPR crystals (Fig. 2 ▶ a) belong to space group P2₁2₁2₁, with unit-cell parameters a = 89.7, b = 104.0, c = 138.4 Å. The Matthews coefficient (Matthews, 1968 ▶) of 2.22 ų Da⁻¹ and solvent content of 44.69% assume the presence of four subunits of MtbAGPR per asymmetric unit. A tetramer is the active biological unit in related structures from other organisms (PDB codes 1vkn and 2cvo) determined to date. The program MOLREP (Vagin & Teplyakov, 1997 ▶) from the CCP4 suite of programs (Collaborative Computational Project, Number 4, 1994 ▶) was used to generate a solution for MtbAGPR using the atomic coordinates of PDB entry 1vkn (T. maritima, 38% identity) as a search model. This solution confirmed the presence of four subunits in the asymmetric unit.

Figure 2.

Diffraction-quality crystals of each enzyme. (a) MtbAGPR of approximate maximum dimensions of 200 × 20 × 20 µm grown in 0.1 M bis-tris, 20% PEG MME 5000 pH 6.5. (b) MtbOTC crystals grown in 0.1 M MES, 1.6 M magnesium sulfate pH 6.5. The approximate average crystal dimensions are 50 × 50 × 50 µm.

The tetragonal MtbOTC crystals (Fig. 2 ▶ b; space group P4₁2₁2) have unit-cell parameters a = b = 136.1, c = 141.7 Å. The Matthews coefficient for three molecules in the asymmetric unit is 3.31 ų Da⁻¹, corresponding to a solvent content of 62.87%. Other OTC crystal structures determined to date (PDB codes 1akm and 1fb5) have a trimer as the active biological unit. The MtbOTC asymmetric unit contains density for three subunits as determined by molecular replacement using PDB entry 1fb5 as a search model (ovine OTC, 38% identity). Crystallographic refinement and molecular-structure analysis for both of these enzymes will be published elsewhere in the future.