Identification, cloning and functional expression of the gene encoding OMP decarboxylase from Plasmodium falciparum
Key words: OMP decarboxylase, pyrimidine, Plasmodium, drug target, Malarial Genome
R. Ian Menz1, Olivier Cinquin and Richard I. Christopherson*
School of Molecular and Microbial Biosciences The University of Sydney, Sydney, NSW 2006, AUSTRALIA
* Address for correspondence:School of Molecular and Microbial Biosciences, The University of Sydney, Sydney, NSW 2006, AUSTRALIA, Phone: 61 2 9351 6031 Fax: 61 2 9351 4726, Email: ric@mmb.usyd.edu.au
1 Current address: School of Biological Sciences, The Flinders University of South Australia Bedford Park, SA 5042, AUSTRALIA
Original manuscript, published in Ann. Trop. Med. Parasitol. 96(5), pp469-476 (2002)
Abstract
The coding region of a putative orotidine 5-monophosphate decarboxylase gene from Plasmodium falciparum was identified in genomic data from the Malarial Genome Sequencing Project. The gene encodes a protein of 323 amino acids with a predicted molecular weight of 37.8 kDa. The gene was cloned into a bacterial expression vector and over-expressed in Escherichia coli. The recombinant protein was purified and shown to have orotidine 5-monophosphate decarboxylase activity, confirming the identity of the gene.
Abbreviations: ODCase, OMP decarboxylase; OMP, orotidine 5-monophosphate; OPRTase, orotate phosphoribosyltransferase.
Introduction
The malarial parasite, Plasmodium falciparum, is the major causative agent of human malaria (Winstanley, 2000). The emergence of drug-resistant strains (Winstanley, 2000) has increased the demand for potent new anti-malarial drugs with different mechanisms of action. The enzymes of the de novo pyrimidine pathway are ideal targets for antimalarial drugs. The human host has alternative salvage pathways for synthesis of pyrimidine nucleotides, but the malarial parasite is solely reliant on the de novo pathway (Scheibel and Sherman, 1988) for synthesis of DNA and RNA and cell division. Anti-malarial drugs which target this pathway do not need absolute specificity for the parasitic de novo enzymes, as the host can salvage pyrimidine nucleotides from precursors in the blood such as uridine.
Orotidine 5-monophosphate decarboxylase (ODCase, EC 4.1.1.23 ) catalyzes the sixth step of the de novo pyrimidine pathway, the decarboxylation of OMP to uridine 5-monophosphate (OMP -> UMP; Jones, 1980). In mammals and other multicellular eukaryotes, ODCase resides on a bifunctional protein which also contains orotate phosphoribosyltransferase (OPRTase; Yablonski et al., 1996). In lower eukaryotes and prokaryotes, these two enzymes are distinct and mono-functional. ODCase has not been purified to homogeneity from P. falciparum; however, the ODCase and OPRTase activities have been chromatographically resolved demonstrating that these enzymes are distinct in P. falciparum (Rathod and Reyes, 1983). The mono-functional ODCases are active as homo-dimers with subunit molecular weights ranging from 22.8 to 43.9 kDa (Glazebrook et al., 1987; Nelson et al., 1999). Unlike other decarboxylases, ODCase does not utilise metals or other cofactors and is an extremely proficient enzyme, enhancing the rate of spontaneous decarboxylation by 17 orders of magnitude (Miller et al., 1999). There has been much debate over the reaction mechanism; however, recently, the structures of ODCase from several species have been solved, and all suggest similar mechanisms (Appleby et al., 2000; Harris et al., 2000; Miller et al., 2000; Wu et al., 2000).
The increasing world-wide problem caused by malaria has lead to establishment of the Malaria Genome Sequencing Project, a consortium of three institutions devoted to the complete sequencing of the P. falciparum genome. The availability of this sequence da ta will expedite investigation of malarial proteins and provide new targets for drug design. Due to the technical difficulties in obtaining large quantities of pure enzyme from parasites grown in erythrocytic culture, many potential drug targets have not been extensively characterised. The availability of genomic data has enabled the cloning, expression and purification of potential drug targets in milligram quantities enabling full characterization of interactions with inhibitors and determination of th ree-dimensional structures using X-ray diffraction and NMR spectroscopy.
2. Materials and methods
2.1 DNA cloning
The coding region of the putative ODCase gene, identified by homology with sequences from other species, was amplified by PCR from P. falciparum (strain 3D7) genomic DNA using a mixture of Taq and Vent polymerases with the primers PFODCF (5-GTATACGCGTATCGAAGGTCGTATGGGTTTTAAGGTAAAATTAGAA AAACGAAGGAATGC-3) and PFODCR (5-GTCCACCATGGTTACGATTCCATATTTTGCTTTAAGATTGCATTAATCTGATCG-3). PFODCF inc orporated sequence that encoded a Factor Xa protease cleavage site (double underlined) to facilitate removal of the N-terminal His tag. Reactions were cycled with the following parameters: 94 deg C for 1 min, 46 deg C for 1 min and 5 min at 65 deg C for 30 cycles; 65 deg C for 10 min. The resultant fragment was sub-cloned into the expression vector pETMCSIII (Neylon et al., 2000) using Mlu I and NcoI restriction sites (underlined) incorporated into PFODCF and PFODCR, respectively, to yield the construct pETMCSIII-ODC. A second expression construct was synthesised by amplifying the ODCase gene from the pETMCSIII-ODC construct using the primers PFODC3F (5- gtccaccATGGGTTTTAAGGTAAAATTAGAAAAACGAAGGAATGC-3) and PFODC3R (5-cagccggatccttaatggtgatggtgatggtgatggtgatggctgccgcgcggcaccaggGATTCCATATTTTGCTTTAAGATTGCATTAATCTGATCG-3) and the same PCR protocol. PFODC3R incorporated sequence for a thrombin protease cleavage site (double underlined) and a 9 x C-terminal His tag. The resultant fragment was sub-cloned into pET-3d (Novagen, Madison, WI, USA) using the NcoI and BamHI restriction sites (underlined) incorporated into PFODC3F and PFODC3R, respectively, to yield the expression construct pET3d-ODC3.
2.2 Over-expression and enzyme purification
E. coli strain BL21(DE3) (Novagen, Madison, WI, USA) was transformed with the expression construct, pET3d-ODC3, and the pMICO plasmid encoding 3 rare tRNAs and T7 lysozyme (Cinquin et al., 2001). Large-scale cultures were grown in a New Brunswick BioFlow III fermentor (New Brunswick Scientific, Edison, NJ, USA) containing 4-5 l of L-broth (Sambrook et al., 1989) with glucose (0.1% (w/v)), ampicillin (100 micro-g/ml) and chloramphenicol (100 micro-g/ml). Cultures were grown at 37 deg C until the optical density at 600 nm reached approximately 1.2, the temperature was then reduced to 25 deg C, and protein expression induced by addition of IPTG (250 micro-M). The culture was grown overnight and the cells were harvested by centrifugation (4000 x g, 10 min, 4 deg C). The cell pellet was resuspended in Ni-NTA buffer (30 ml/l of culture) containing 50 mM Tris.HCl pH 8.0, 300 mM NaCl and 5 mM imidazole and the cells were disrupted by two passages at 10,000 psi through a Rainne lab homogeniser (APV homogenisers, Tempress, Denmark). The cell extract was centrifuged (38,000 g, 30 min, 4o C), the supernatant filtered through a 0.45 micro-m filter and applied to a 2.5 ml Ni-NTA agarose column (Qiagen, Valencia, CA, USA) pre-equilibrated with Ni-NTA buffer. The column was washed with Ni-NTA buffer containing 20 or 60 mM imidazole for the 6- or 9-His tagged proteins, respectively, until no protein was detected in the eluate. The recombinant ODCase was then eluted from the column with Ni-N TA buffer containing 250 mM imidazole. The ODCase was dialysed overnight at 4 deg C against protease cleavage buffer containing 20 mM Tris-HCl pH 8.5, 200 mM NaCl, 5 mM CaCl2 and 10% (v/v) glycerol. The His tag was removed by incubation (20o C, 16 h) with 0.06 U of either thrombin or Factor Xa, per mg protein, proteolysis was terminated by addition of PMSF (1 mM).
2.3 Characterization of recombinant protein
ODCase activity was measured at room temperature in 25 mM MOPS pH 7.0 and 100 mM NaCl. The reaction rate was calculated from the decrease in absorbance at 285 nm where delta-epsilon = - 1650 cm-1 M-1 (Livingstone and Jones, 1987).
Protein concentration was determined by the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). SDS-PAGE electrophoresis was carried out according to Laemmli, (1970). Mass spectrometry was performed with a Finnigan LCQ (Thermoquest, San Jose, CA, USA) operating in positive ion, ESI mode. Samples were introduced to the mass spectrometer after chromatography on a Jupiter C18 column (Phenomenex, Torrance, CA, USA) using a linear gradient of 2 to 98 % acetonitrile in 0.02% (v/v) TFA and 0.1 % (v/v) acetic acid.
3. Results and discussion
3.1 Identification and cloning of the orotidine 5-monophosphate decarboxylase gene
The amino acid sequences for ODCases from 74 species were obtained from the Swissprot database and aligned using ClustawlX (Thompson et al., 1997). Figure 1 shows alignment of some of these sequences. Ten residues were totally conserved (6 identical) and a further 36 residues were 80% conserved among all 74 species (Fig. 1). The consensus sequence FEDRKFADIGNTV for a highly conserved region was identified (Fig. 1). If a residue was not completely conserved among the 74 species, then the amino acid that occurred with the highest frequency was used in that position. The consensus sequence was then used to tBLASTn search preliminar y sequence data from the Malaria Genome Sequencing Project http://www.ncbi.nlm.nih.gov/Malaria). Only one sequence provided by The Institute for Genomic Research (TIGR, Temp_id 2154) was identified with an open reading frame of sufficient size to encode ODCase. This DNA nucleotide sequence was translated into an amino acid sequence and aligned with ODCase sequences from the other 74 organisms (Fig. 1). This sequence contained all the completely conserved residues, and 17 of the 36 residues that were 80% conserved amongst the other 74 species and therefore represented a putative ODCase gene. The putative gene encoded a protein of 323 amino acids with a theoretical molecular weight of 37,824 Da, within the range of 22.8 to 43.9 kDa (Glazebrook et al., 1987; Nelson et al., 1999) reported for other mono-functional ODCases.
PCR primers were designed to amplify the coding region from P. falciparum strain 3D7 genomic DNA and to incorporate restriction enzyme sites suitable for cloning into pETMSCIII (Neylon et al., 2000) which adds an N-terminal 6 x His tag. The primers also introduced a Factor Xa protease cleavage site between the His tag and ODCase. Only a single PCR product of approximately 1,000 bp was observed with these primers (Fig. 2), consistent with that predicted from the gene sequence. The PCR product was cloned into pETMSCIII (Neylon et al., 2000) and the sequence of the cloned fragment (GenBank accession no. AF462064) was identical to the preliminary sequence from TIGR. Subsequently, TIGR placed this sequence into a preliminary assemblage of chromosome 10, assigned it Temp_id 345.t00008, and putatively identified the gene as OMP decarboxylase (http://www.tigr.org).
3.2 Over-expression and purification of recombinant ODCase
Initial over-expression trials with the pETMCSII-ODC vector were p erformed with BL-21 (DE3) CodonPlus-RIL cells (Stratagene, La Jolla, CA, USA) or BL21 (DE3) (Novagen, Madison, WI, USA) carrying the RIG plasmid (Baca and Hol, 2000) to overcome the codon bias of the malarial ODCase gene. The malarial ODCase gene appeared to be toxic to these recipient strains of E. coli. Therefore, the pMICO plasmid, which encodes the tRNA genes, argU (AGA/AGG), ileX (ATA) and glyT (GGA), and T7 lysozyme was developed and is described elsewhere (Cinquin et al., 2001).
The pMICO plasmid facilitated the over-expression of ODCase (Cinquin et al., 2001). However the 6 x His tag did not provide sufficiently strong binding to the nickel affinity column, the tagged ODCase started to elute at imidazole concentrations greater than 20 mM (not shown). This low stringency washing yielded substantial amounts of ODCase with several contaminants (not shown). This partially pure ODCase was treated with Factor Xa and passed back through the Ni-NTA column to remove the free tag and any uncleaved protein but no cleaved ODCase protein was detected in the eluate by activity assay or SDS-PAGE (not shown).
The low affinity of the 6xHis tagged ODCase for the Ni-NTA column and the inability of the tag to be cleaved by Factor Xa suggested that the tertiary structure of the N-terminus of the recombinant protein may obscure the Factor Xa site and part of the 6x His tag. Therefore, a new expression construct containing a thrombin cleavage site and 9 x His tag at the C-terminus of the protein was constructed. This construct expressed ODCase to similar levels in the BL21 pMICO cells (not shown). The 9 x His tag had a greater affinity for the nickel affinity column and could be washed with up to 60 mM imidazole without eluting the ODCase (Fig. 2). Following washing, essentially pure ODCase, with an apparent molecular weight of 35 kDa, was eluted with 250 mM imidazole (Fig. 2). After thrombin digestion, the majority of the ODCase activity passed through the Ni-NTA agarose column without binding and a slight reduction in the apparent size was observed by SDS-PAGE, consistent with removal of the 9 x His tag.
3.3 Characterisation of recombinant malarial ODCase
The molecular weight of the cleaved ODCase estimated by SDS- PAGE appeared slightly smaller than the 38,289 Da predicted from the sequence. The molecular weight of the product was determined as 38,158 Da by mass spectrometry, consistent with the cleaved protein after removal of the start methionine (residue m.w. 131).
The specific enzymic activity of the cleaved ODCase determined at various concentrations was 7.5 mic-mol UMP min-1 mg protein-1. A similar specific activity was also observed for the precursor 9 x His tagged enzyme. This specific activity equates to a turn-over number for the P. falciparum ODCase of 5 s-1, slightly lower than the turn-over numbers of pure enzymes from other species, but of a similar order of magnitude (Table 1). The turn-over number for the P. falciparum ODCase was determined under assays conditions similar to those used for the yeast enzyme (Miller et al., 1999).
Acknowledgments We thank Drs. Alan Cowman and Mark Wickham (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) for providing P. falciparum, 3D7 genomic DNA and Prof. Wim Hol and Dr. Arthur Baca (Howard Hughes Medical Institute, University of Washington, Seattle, USA) for providing the RIG plasmid. We thank Hiu Chuen Lok for assistance with some experiments. Preliminary s equence data for P. falciparum chromosomes 10 and 11 were obtained from The Institute for Genomic Research website (www.tigr.org). Sequencing of chromosomes 10 and 11 was part of the International Malaria Genome Sequencing Project and was supported by awa rd from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
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Figure 1 (see PDF document). Alignment of amino acid sequences of ODCases from selected organisms. The sequences from 74 ODCase genes were aligned and a sub-set is presented here. Identical residues are shaded black, residues that are 100% and 80% conserved amongst the 74 sequences are shaded dark and light grey, respectively. The consensus amino acid sequence used to identify the putative P. falciparum gene is shown above. Species abbreviated above are Plasmodium falciparum, Drosophila melanogaster, Dictyostelium discoideum, Saccharomyces cerevisiae , Pseudomonas aeruginosa, Pyrococcus horikoshii and Methanobacterium thermoautotrophicum. * Bifunctional protein.
Figure 2. Analysis of DNA and Protein for P. falciparum ODCase. Panel A: Lane 1, reaction product from PCR with primers PFODC3F and PFODC3R, the size and migration of DNA standards are indicated. Panel B: Lane 1, contaminating proteins eluted from Ni-NTA agarose with 60 mM imidazole; Lane 2, purified ODCase eluted from Ni-NTA with 250 mM imidazole; Lane 3, ODCase following thrombin cleavage and Ni-NTA purification. The sizes of protein standards are indicated.