Crystallization and preliminary X-ray crystallographic analysis of human quinolinate phosphoribosyltransferase

H. sapiens quinolinate phosphoribosyltransferase has been expressed, purified and crystallized. A diffraction data set has been collected and processed at 2.8 Å resolution.

Abstract

Quinolinate phosphoribosyltransferase (QPRTase) is a key NAD-biosynthetic enzyme which catalyzes the transfer of quinolinic acid to 5-phosphoribosyl-1-pyrophosphate, yielding nicotinic acid mononucleotide. Homo sapiens QPRTase (Hs-QPRTase) appeared as a hexamer during purification and the protein was crystallized. Diffraction data were collected and processed at 2.8 Å resolution. Native Hs-QPRTase crystals belonged to space group P2₁, with unit-cell parameters a = 76.2, b = 137.1, c = 92.7 Å, β = 103.8°. Assuming the presence of six molecules in the asymmetric unit, the calculated Matthews coefficient is 2.46 ų Da⁻¹, which corresponds to a solvent content of 49.9%.

1. Introduction

Quinolinate phosphoribosyltransferase (QPRTase) is an essential enzyme for the de novo biosynthesis of nicotinamide adenine di­nucleotide (NAD) in both prokaryotes and eukaryotes (Foster & Moat, 1980 ▶). This enzyme catalyzes the formation of nicotinic acid mononucleotide (NAMN), pyrophosphate and CO₂ from quinolinic acid (QA) and 5-phosphoribosyl-1-pyrophosphate (PRPP) in a reaction that involves phosphoribosyl transfer followed by decarboxylation. QA is the first intermediate in this biosynthetic process. In eukaryotes QA is produced via degradation of tryptophan, while in prokaryotes it is produced from dihydroxylacetone phosphate and l-­aspartate. Notably, QA is a known neurotoxin in mammals: its action is exerted via sustained activation of glutamate N-­methyl-d-­aspartate receptors and it is involved in a variety of neurological diseases (Foster et al., 1985 ▶; Foster & Schwarcz, 1985 ▶; Feldblum et al., 1988 ▶).

The structures of QPRTase from Homo sapiens, Saccharomyces cerevisiae, Helicobacter pylori, Thermus thermophilus, Mycobacterium tuberculosis, Salmonella typhimurium and Thermotoga maritima have all been shown to consist of an N-terminal four-stranded open-face β-sandwich domain and a C-terminal α/β-barrel domain (Eads et al., 1997 ▶; Sharma et al., 1998 ▶; Schwarzenbacher et al., 2004 ▶; Kim et al., 2006 ▶; Liu et al., 2007 ▶; di Luccio & Wilson, 2008 ▶). QPRTases exist as dimers or hexamers; those from eukaryotes, including yeast, hog, rat and human, are hexamers in solution (Iwai & Taguchi, 1974 ▶; Okuno & Schwarcz, 1985 ▶; Okuno et al., 1988 ▶; di Luccio & Wilson, 2008 ▶). Consistent with these findings, structural studies of QPRTases from Homo sapiens and Saccharomyces cerevisiae showed that they form a hexameric structure (Liu et al., 2007 ▶; di Luccio & Wilson, 2008 ▶). In contrast, most QPRTases from prokaryotes, including those from M. tuberculosis, Salmonella typhimurium and Thermotoga maritima, are dimeric, although some prokaryotic QPRTases (those from Helicobacter pylori and Thermus thermophilus) have a hexameric structure identical to the enzymes from Homo sapiens and Saccharomyces cerevisiae.

The reaction catalyzed by QPRTase is central and indispensable to NAD biosynthesis and cell survival in prokaryotes. Thus, QPRTases from bacterial pathogens such as Helicobacter pylori, M. tuberculosis and S. typhimurium represent attractive targets for antibacterial drugs (Eads et al., 1997 ▶; Sharma et al., 1998 ▶; Kim et al., 2006 ▶) and structural information about the host QPRTase is essential for the proper design of specifically antibacterial inhibitors. Currently, only the structure of Homo sapiens QPRTase (Hs-QPRTase) in complex with a tartrate molecule, which partly mimics QA, is available (Liu et al., 2007 ▶). Once we know the apo structure of Hs-QPRTase, we should be able to determine the mechanism underlying substrate recognition and provide structural information for drug development. As a first step towards this goal, we report here the overexpression, purification and crystallization of apo Hs-QPRTase and its preliminary X-ray characterization.

2. Materials and methods

2.1. Expression and purification

DNA encoding Hs-QPRTase (residues 1–297) was amplified by PCR and subcloned into the NdeI and XhoI sites of the expression vector pET-21a (Novagen). After transformation into Escherichia coli strain C41 (DE3), recombinant Hs-QPRTase was induced by treating the cells with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 6 h at 310 K. The cells were then harvested and resuspended in lysis buffer containing 50 mM sodium phosphate pH 7.5, 300 mM NaCl, after which the suspended cells were lysed by sonication and centrifuged at 14 000g for 1 h. The supernatant was collected and loaded onto a column packed with Ni–NTA resin (Peptron) pre-equilibrated with lysis buffer. After washing with wash buffer (50 mM sodium phosphate pH 7.5, 300 mM NaCl, 10 mM imidazole), the bound protein was eluted with elution buffer (50 mM sodium phosphate pH 7.5, 300 mM NaCl, 250 mM imidazole). The Hs-QPRTase protein was then further purified by size-exclusion chromatography on a Superdex 200 column (Pharmacia) equilibrated with 20 mM HEPES–NaOH pH 7.5, 100 mM KCl. The fractions containing the recombinant protein were pooled and concentrated to 15 mg ml⁻¹ using a Centriprep-10 (Amicon).

2.2. Crystallization and data collection

Full-length Hs-QPRTase (residues 1–297), supplemented with an additional Leu and Glu and a hexahistidine tag at its C-terminus, was crystallized at room temperature (294 ± 1 K) using the hanging-drop vapour-diffusion method. The crystals were grown on a siliconized cover slip by equilibrating a mixture consisting of 1 µl protein solution (15 mg ml⁻¹ protein in 20 mM HEPES–NaOH pH 7.5, 100 mM KCl) and 1 µl reservoir solution [100 mM MES–NaOH pH 5.0, 7–15%(w/v) PEG MME 2K, 10 mM KSCN] against 500 µl reservoir solution. Plate-like single crystals grew to maximum dimensions of 0.3 × 0.3 × 0.1 mm over the period of a week (Fig. 1 ▶). They were cryoprotected in reservoir solution supplemented with 25%(v/v) glycerol and flash-cooled under N₂ gas at 95 K. Native data were collected to 2.8 Å resolution from a single frozen crystal using an ADSC Quantum 210 CCD detector on beamline NW12 of Photon Factory (Tsukuba, Japan). The data set was processed and scaled using HKL-2000 (Otwinowski & Minor, 1997 ▶). The data-collection statistics are summarized in Table 1 ▶.

Figure 1.

Native crystals of Hs-QPRTase grown for 7 d in 100 mM MES–NaOH pH 5.0, 7–12%(w/v) PEG MME 2K, 10 mM KSCN. The crystal dimensions are approximately 0.3 × 0.3 × 0.1 mm.

Table 1. Data-collection statistics.

Values in parentheses are for the highest resolution shell.

X-ray source	PF-AR NW12
Wavelength (Å)	1.0000
Space group	P21
Unit-cell parameters (Å, °)	a = 76.2, b = 137.1, c = 92.7, β = 103.8
Resolution range (Å)	50–2.8 (2.9–2.8)
Observed reflections	196103
Unique reflections	42775
Multiplicity	4.7 (4.0)
Completeness (%)	97.3 (94.2)
Rmerge† (%)	6.4 (31.6)
〈I/σ(I)〉	18.3 (4.8)

^† R _merge = , where I _i(hkl) is the intensity of the ith observation of reflection hkl and 〈I(hkl)〉 is the average intensity of reflection hkl.

3. Results and discussion

The Hs-QPRTase crystals belonged to space group P2₁, with unit-cell parameters a = 76.2, b = 137.1, c = 92.7 Å, β = 103.8°. Assuming the presence of six molecules in the asymmetric unit, the calculated Matthews coefficient is 2.46 ų Da⁻¹, which corresponds to a solvent content of 49.9% (Matthews, 1968 ▶). Molecular-replacement calculations were carried out with MOLREP (Vagin & Teplyakov, 2010 ▶) using the structure of the Hs-QPRTase–tartrate complex (PDB code 2jbm; Liu et al., 2007 ▶) as a search model. The cross-rotation function for this model was calculated using data in the resolution range 15–4 Å and the first six peaks in the output appeared as distinct solutions. These six peaks grew more distinct in the translation calculations, with an overall correlation coefficient of 54.0% and an R factor of 48.2%. After refinement using the program CNS (Brünger et al., 1998 ▶), the resultant electron-density map showed six molecules in an asymmetric unit and the quality of the initial map was high enough to build most of the residues. Model building and further refinement are ongoing.