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Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 September 1; 64(Pt 9): 805–808.
Published online 2008 August 9. doi:  10.1107/S1744309108024500
PMCID: PMC2531282

Expression, purification and preliminary crystallographic analysis of N-acetylglucosamine-1-phosphate uridylyltransferase from Mycobacterium tuberculosis

Abstract

The gene product of open reading frame Rv1018c from Mycobacterium tuberculosis is annotated as encoding a probable N-acetylglucosamine 1-­phosphate uridylyltransferase (MtbGlmU), an enzyme that catalyzes the biosynthesis of UDP-N-acetylglucosamine, a precursor common to lipopolysaccharide and peptidoglycan biosynthesis. Following overexpression in Escherichia coli, the enzyme was purified and crystallized using the hanging-drop vapour-diffusion method. Native diffraction data were collected from crystals belonging to space group R32 and processed to a resolution of 2.2 Å.

Keywords: Mycobacterium tuberculosis H37Rv, Rv1018c, N-acetylglucosamine 1-phosphate uridyltransferase, peptidoglycan metabolism, GlmU

1. Introduction

Discovered more than a century ago by Robert Koch, Mycobacterium tuberculosis is still a leading cause of human casualties resulting from infectious diseases. More than two billion people are currently infected with M. tuberculosis, with new cases accumulating at a rate of nine million per annum. In 2006, 1.7 million people died from tuberculosis alone or in combination with AIDS, according to a report by the World Health Organization (WHO; http://www.who.int/tb).

Despite an initial period of decline in the incidence of tuberculosis (TB) since the introduction of the BCG vaccine and anti-TB drugs, the disease has been resurgent in the past few decades. In 1993, tuberculosis was declared to be a global emergency by the WHO. This resurgence of TB can be linked to several factors. Firstly, HIV infection has created a large highly susceptible population of individuals for whom TB is the most common cause of death owing to their compromised immune systems (Nunn et al., 2005 [triangle]). Furthermore, noncompliance with existing anti-TB therapies has given rise to multi-drug-resistant and extensively drug-resistant M. tuberculosis strains, further compounding the already grave situation (Matteelli et al., 2007 [triangle]). The M. tuberculosis Structural Genomics Consortium was formed in 2000 (http://www.doe-mbi.ucla.edu/TB) with the goal of providing structural bases for the development of novel effective therapeutics for tuberculosis (Goulding et al., 2002 [triangle]; Smith & Sacchettini, 2003 [triangle]; Terwilliger et al., 2003 [triangle]; Murillo et al., 2007 [triangle]). Such a systematic approach is possible as the complete genome of the best-characterized virulent strain of M. tuberculosis, H37Rv, has been sequenced and many of its open reading frames have been annotated (Camus et al., 2002 [triangle]; Cole et al., 1998 [triangle]).

In this study, we report the molecular cloning, protein purification and preliminary X-ray crystallographic analysis of the Rv1018c gene product, which has been annotated as a probable N-acetyl­glucosamine 1-phosphate uridyltransferase (EC 2.7.7.23; MtbGlmU). GlmU is a bifunctional enzyme that catalyzes two consecutive reactions leading to the biosynthesis of UDP-N-acetylglucosamine (UDP-GlcNAc). In the first reaction, GlmU catalyzes the CoA-dependent acetylation of glucosamine-1-phosphate (Glc-1-P) to afford N-acetyl­glucosamine-1-­phosphate (GlcNAc-1-P). The acetyltransferase domain resides in the C-terminal ~230 residues of GlmU (253–456 in Escherichia coli GlmU numbering), which form a very long left-handed β-helix (LβH) domain. In the second reaction, the UMP moiety from a UTP molecule is transferred to GlcNAc-1-P to give the final product UDP-GlcNAc (Gehring et al., 1996 [triangle]). The N-terminal uridyltrans­ferase domain (residues 1–250 in E. coli GlmU num­bering) displays a Rossmann-like fold with a central seven-stranded β-sheet surrounded by six helices (Brown et al., 1999 [triangle]; Gehring et al., 1996 [triangle]). The acetyltransferase and the uridyltransferase domains have been separately expressed and purified; they both showed their corresponding catalytic activities in the absence of the other domain of the full-length enzyme (Olsen & Roderick, 2001 [triangle]).

2. Experimental methods

2.1. Cloning, expression and purification

The template DNA for Rv1018c amplification by the polymerase chain reaction (PCR) was obtained from a bacterial artificial chromosome (BAC) genomic library of the M. tuberculosis H37Rv strain from L’Institut Pasteur (Brosch et al., 1998 [triangle]; Gordon et al., 1999 [triangle]). The PCR primer sequences (Integrated DNA Technologies) for directional cloning of the amplified product into the Gateway cloning system (Invitrogen) were Rv1018cF (5′-GGGACAAGTTTGTACAAAAAAGCAGGCTCCGAAAACCTGTATTTTCAGGGATGACGTTTCCTGGTGACACCGCG-3′) and Rv1018cR (5′-GGGACCACTTTGTACA­AGAAAGCTGGGTC-3′). The bold type in the forward primer indicates the nucleotides encoding the cleavage site (ENLYFQ^G) for tobacco etch virus protease (TEVpro). Complete proteolysis by TEVpro leaves a single glycine residue at the amino-terminus as indicated. The PCR product containing the full open reading frame encoding residues 1–495 of Rv1018c was gel-purified and inserted into the pDONR221 plasmid (Invitrogen) to generate an entry clone plasmid pDONR1018c. Subsequently, the coding region of Rv1018c in pDONR1018c was cloned into expression plasmid pDEST-15 (Invitrogen) with an amino-terminal glutathione S-transferase fusion partner. The coding sequence of Rv1018c in this expression plasmid (pGST-1018c) was confirmed by DNA-sequencing analysis (DNA Core Facility, Department of Biochemistry, University of Alberta, Canada).

The expression strain E. coli BL21 (DE3) (Novagen) was transformed to ampicillin resistance by pGST-1018c. An overnight culture from a single colony was used to inoculate 2 l Terrific Broth (TB) supplemented with 100 µg ml−1 ampicillin. Shaking of the culture at 310 K was continued until its OD600nm reached 0.8. Subsequently, the temperature of the culture was shifted to 295 K and protein overexpression was induced by adding isopropyl β-d-1-thiogalacto­pyranoside (IPTG) to a final concentration of 0.5 mM. After overnight incubation, the cells were harvested by centrifugation at 7000g for 15 min. Bacterial pellets were resuspended in 40 ml buffer A (20 mM NaH2PO4/Na2HPO4 pH 7.4, 300 mM NaCl, 1 mM DTT) supplemented with a tablet of Complete protease-inhibitor cocktail (Roche). For protein purification, the cells were lysed by freeze–thawing and then subjected to ultrasonication in buffer A. The lysate was cleared by centrifugation (30 min, 20 000g) and the supernatant was loaded onto a 5 ml GSTrap glutathione Sepharose column (GE Healthcare) pre-equilibrated with phosphate-buffered saline (PBS). The GST-Rv1018c fusion protein was competitively eluted from the column with 10 mM reduced glutathione (Sigma) in 50 mM Tris–HCl pH 8.0, 2 mM DTT and 0.02% NaN3. The GST tag was removed by proteolytic cleavage using recombinant tobacco etch virus protease (AcTEV; Invitrogen). The site recognized by the AcTEV protease is encoded in the forward primer (shown in bold in the sequence above) and leaves a single glycine residue in the P1′ position following complete proteolysis. After overnight dialysis against PBS, the cleaved protein mixture was once again loaded onto a GSTrap column and the flowthrough fractions containing liberated Rv1018c were dialyzed against 10 mM Tris–HCl pH 7.4 and 100 mM NaCl. The resulting solution was concentrated to ~12 mg ml−1 using an Amicon Ultra filtration unit (5 kDa cutoff; Millipore). All steps of protein purification subsequent to the initial thawing of cells were performed at 277 K and the result of each step was monitored by 15% SDS–PAGE.

2.2. Crystallization

Initial screening of commercially available crystallization conditions was performed on native full-length Rv1018c at 295 K using the sitting-drop vapour-diffusion technique in 96-well Intelliplates (Hampton Research). Crystal Screens I and II and Index Screen (Hampton Research) were applied using equal volumes (0.2 µl) of protein solution and reservoir solution. Periodic checking of the crystallization setups over four months indicated that Rv1018c crystals grew under five conditions, with the largest crystals observed in drops containing Index Screen condition No. 91. To obtain crystals suitable for X-ray diffraction, equal volumes (1 µl) of protein solution at ~10 mg ml−1 and reservoir solution (20% PEG 3350, 0.15 M dl-­malate pH 7.0) were mixed and equilibrated in hanging drops against 1 ml reservoir solution on a 24-well VDX plate (Hampton Research). Crystals grew to maximal dimensions of 40 × 50 × 50 µm over two weeks (Fig. 1 [triangle]).

Figure 1
Morphology of Rv1018c crystals. The approximate dimensions of Rv1018c crystals of diffraction quality grown in 20% PEG 3350, 0.15 M dl-malate pH 7.0 over a period of two weeks were 40 × 50 × 50 µm. ...

2.3. Data collection

Crystals for synchrotron data collection were first rinsed in cryoprotectant (30% glycerol in reservoir solution) and then flash-cooled in liquid nitrogen. Native data sets were collected on beamline CMCF1 at the Canadian Light Source (CLS). Raw data were indexed, integrated and scaled using the HKL-2000 program suite (Otwinowski & Minor, 1997 [triangle]).

3. Results

Native data sets (Fig. 2 [triangle]) were collected from Rv1018c crystals on beamline CMCF1 of the Canadian Light Source. Crystal parameters and crystallographic statistics of the data sets are summarized in Table 1 [triangle]. According to the calculated molecular weight of Rv1018c including the N-terminal glycine as the result of AcTEV cleavage, 51 641.05 Da, the Matthews coefficient (Matthews, 1968 [triangle]) is calculated to be 2.4 Å3 Da−1 with a solvent content of 48.1%. This result corresponds to the presence of one MtbGlmU molecule per asymmetric unit. The biological assembly of GlmU enzymes from other bacterial species is thought to consist of a homotrimer related by threefold symmetry (Sulzenbacher et al., 2001 [triangle]; Brown et al., 1999 [triangle]; Mochalkin et al., 2007 [triangle]). The available crystal structures of GlmU enzymes show only one (Kostrewa et al., 2001 [triangle]; Mochalkin et al., 2007 [triangle]) or two (Sulzenbacher et al., 2001 [triangle]; Olsen & Roderick, 2001 [triangle]) molecules of GlmU in the asymmetric unit. However, the crystal symmetry of these structures always allows the reconstruction of a GlmU homotrimer via the symmetry operations. Most of the inter-protomer interactions within the GlmU trimer are provided by the C-terminal LβH domain. Based on the significant sequence conservation in the C-terminal acetyltransferase domains of MtbGlmU and other bacterial GlmU enzymes (Fig. 3 [triangle]) and the finding that the MtbGlmU crystals belong to space group R32, we posit that MtbGlmU also assembles into a crystallographic homotrimer.

Figure 2
A representative frame showing the diffraction quality of Rv1018c crystals. The boxed area is enlarged and shown on the right; numbers indicate resolutions in Å.
Figure 3
Sequence alignment of MtbGlmU with GlmU enzymes from Streptococcus pneumonia (SpGlmU), Haemophilus influenza (HiGlmU) and E. coli (EcGlmU). The numbering of the alignment corresponds to the sequence of Rv1018c from M. tuberculosis. The secondary structures ...
Table 1
Crystal parameters and data-collection statistics for native Rv1018c

Acknowledgments

X-ray diffraction data were collected on beamline CMCF1 at the Canadian Light Source. Research in the laboratory of MNGJ is funded by the Alberta Heritage Foundation for Medical Research (AHFMR) and the Canadian Institute of Health Research (CIHR). MNGJ holds a Canada Research Chair in Protein Structure and Function. JY is grateful for the support of a postdoctoral fellowship from AHFMR.

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Articles from Acta Crystallographica Section F: Structural Biology and Crystallization Communications are provided here courtesy of International Union of Crystallography