Molecular and biochemical characterization of nematode cofactor independent phosphoglycerate mutases

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Molecular & Biochemical Parasitology 156 (2007) 210–216

Molecular and biochemical characterization of nematode cofactor independent phosphoglycerate mutases夽 Sylvine Raverdy, Yinhua Zhang, Jeremy Foster, Clotilde K.S. Carlow ∗ New England Biolabs, Division of Parasitology, 240 County Road, Ipswich, MA 01938, USA Received 11 July 2007; received in revised form 10 August 2007; accepted 13 August 2007 Available online 19 August 2007

Abstract Phosphoglycerate mutase (PGM, EC 5.4.2.1) catalyzes the isomerization of 3-phosphoglycerate and 2-phosphoglycerate in glycolysis and gluconeogenesis. Two distinct types of PGM exist in nature, one that requires 2,3-bisphosphoglycerate as a cofactor (dPGM) and another that does not (iPGM). The two enzymes are structurally distinct and possess different mechanisms of action. In any particular organism, one form may exist or both. Nematodes possess the iPGM form whereas mammals have dPGM. In the present study, we have cloned and expressed iPGM from Onchocerca volvulus and described the catalytic properties of O. volvulus, Brugia malayi and Caenorhabditis elegans iPGM enzymes. Temperature and pH optima were determined for each enzyme. Like other iPGM enzymes, the activities of the nematode iPGM enzymes were dependent on the presence of divalent ions. Inactivation by EDTA could be restored most effectively by magnesium and manganese ions. Kinetic parameters and specific activities of the various recombinant enzymes were determined. The high similarity in catalytic properties among the enzymes indicates that a single enzyme inhibitor would likely be effective against all nematode enzymes. Inhibition of iPGM activity in vivo may lead to lethality as indicated by RNAi studies in C. elegans. Our results support the development of iPGM as a promising drug target in parasitic nematodes. © 2007 Elsevier B.V. All rights reserved. Keywords: Onchocerca volvulus; Brugia malayi; Caenorhabditis elegans; Glycolysis; Gluconeogenesis; Phosphoglycerate mutase

1. Introduction In glycolysis and gluconeogenesis, phosphoglycerate mutase (PGM, EC 5.4.2.1) catalyzes the isomerization of 3phosphoglycerate (3-PG) and 2-phosphoglycerate (2-PG). There are two distinct types of PGM in prokaryotic and eukaryotic organisms, one that requires 2,3-bisphosphoglycerate as a cofactor (dependent PGM or dPGM) and another that does not (independent PGM or iPGM). No amino acid sequence or structural similarities exist between the two enzymes. In any particular organism, one form may be present or both [1,2]. The

Abbreviations: iPGM, cofactor-independent phosphoglycerate mutase; dPGM, cofactor-dependent phosphoglycerate mutase; 2-PG and 3-PG, 2- and 3-phosphoglycerate; Ce, Caenorhabditis elegans; Bm, Brugia malayi; Ov, Onchocerca volvulus; Hs, Homo sapiens 夽 Note: The nucleotide sequence reported in this paper is available in GenBankTM Data Bank under the accession number AY640434. ∗ Corresponding author. Tel.: +1 978 380 7263; fax: +1 978 921 1350. E-mail address: [email protected] (C.K.S. Carlow). 0166-6851/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2007.08.002

two enzymes possess different mechanisms of action. dPGM catalyzes the intermolecular transfer of the phosphoryl group between monophosphoglycerates and the cofactor, with a phosphohistidine as an intermediate [3]. It functions as a monomer (∼250 amino acids), dimer or tetramer [4,5]. In contrast, iPGM catalyzes the intramolecular transfer of the phosphoryl group between the hydroxyl groups of the monophosphoglycerates as a monomer (∼500 amino acids), with a predicted phosphoserine intermediate [6–8]. In addition, dPGM and iPGM can be distinguished by their sensitivity to vanadate and dependence on divalent metal ions. dPGM is inhibited by low concentrations of vanadate while iPGM is not [9]. iPGM enzymes, unlike dPGM, require divalent metal ions for catalysis and the ion of choice for any particular enzyme may differ despite substantial similarity in primary sequence. The iPGMs from many gram-positive bacteria, in particular those from the spore-forming Bacillus species and their close, non-spore forming, relatives have an absolute and specific requirement for manganese ions for catalysis [10,11]. These enzymes have also been shown to be extremely sensitive

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to pH [12]. In the parasitic protozoa Trypanosoma brucei [13] and Leishmania mexicana mexicana [14], cobalt is the preferred divalent metal ion. Differences in amino acid sequence between dPGM, the only PGM present in vertebrates, and nematode iPGM resulted in the identification of Brugia malayi iPGM (Bm-iPGM) as a potential drug target in an in silico screen [15]. An innovative aspect of this method is the use of RNA interference (RNAi) data from the free-living nematode Caenorhabditis elegans to select ‘prevalidated’ drug targets [16]. C. elegans iPGM (Ce-iPGM) and Bm-iPGM were identified in a set of proteins that are absent from mammals yet likely play an important role in nematode biology [15]. Detailed characterization of Ce-iPGM in vivo revealed that the gene is essential in C. elegans because down regulation by RNAi led to embryonic lethality, larval lethality and morphological abnormalities [15]. As expected from its function, the enzyme was expressed in all stages and in many tissues. It is highly likely that the enzyme is equally important in parasitic nematode development. Numerous nematode expressed sequence tags (ESTs) matching iPGM but not dPGM were found, indicating that nematodes possess only one form of PGM [15]. In this study, we demonstrate that the filarial parasite Onchocerca volvulus possesses an active iPGM. We describe the molecular characterization of O. volvulus iPGM (Ov-iPGM), and report on the catalytic properties of two filarial enzymes (Ov-iPGM and Bm-iPGM) and the more distantly related iPGM from C. elegans (Ce-iPGM). The results demonstrate that these enzymes possess similar biochemical characteristics and indicate that a single enzyme inhibitor would likely be effective against all nematode enzymes. 2. Materials and methods 2.1. Identification and cloning of O. volvulus iPGM (Ov-iPGM) B. malayi and C. elegans iPGM amino acid sequences were used to query NCBI databases for an O. volvulus ortholog. Seven matching ESTs derived from various developmental stages (adult worms, infective larvae and microfilariae) were identified. Phage containing inserts corresponding to the ESTs were obtained from the Filarial Genome Project Resource Center ([email protected]) and used as templates for PCR. A specific primer pair was designed corresponding to the putative 5 and 3 ends of the gene: Ov-iPGM-F (5 -ATGAGCGAAGTGAAAAATCGGGT-3 ) and Ov-iPGMR (5 -CTAGACTTCAATAACCACTGG-3 ). Reactions were carried out using 1 ␮M of each primer, 200 ␮M of each dNTP, 0.5 units Vent DNA polymerase (New England Biolabs), 1× ThermoPol Reaction Buffer and the following conditions: 95 ◦ C for 2 min, followed by 25 cycles of 95 ◦ C for 1 min, 58 ◦ C for 1 min, 72 ◦ C for 2 min, and 1 cycle at 72 ◦ C for 10 min. Reaction product corresponding to the full-length ∼1.5 kb insert was obtained and purified. Following the addition of an adenosine single base at the 3 end using Taq DNA polymerase (New England Biolabs) and dATP (New England Biolabs), the insert

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was cloned into pCR2.1-TOPO according to the manufacturer’s instructions (Invitrogen). Plasmid DNA was isolated, and the insert was verified by sequencing. 2.2. Expression and purification of recombinant parasite iPGM and human dPGM enzymes Two primers were designed to subclone the complete O. volvulus iPGM ORF into pET-21a(+) (Novagen) for expression with a 6xHis tag at the C terminus. The forward primer, OviPGMBamHI (5 -ATGCGGATTCATGAGCGAAGTGAAAAATCGGG-3 ) contained a BamHI site (underlined) and the reverse primer, Ov-iPGMXhoI (5 -CGTA CTCGAGGACTTCAATAACCACTGGCTTTCCGG-3 ) contained an XhoI site (underlined). The PCR conditions described above were used. The reaction product was purified, digested with BamHI and XhoI (New England Biolabs) and ligated into pET-21a(+) digested with the same restriction enzymes. The insert was verified by DNA sequencing. A full-length human dPGM (Hs-dPGM, brain form, NM 002629) was amplified from cDNA prepared from the cell line SKNMC. Primers were designed according to the predicted open reading frame: Hs-dPGMBamHI (5 ATAAGTGGATCCATGGCCGCCTACAAACTGGT-3 ) containing a BamHI site (underlined) and Hs-dPGMXhoI (5 -TAAGTTCTCGAGCTTCTTGGCCTTGCCCTG-3 ) containing an XhoI site (underlined). PCR reactions were performed using 1 ␮M of each primer, 200 ␮M of each dNTP, and 2.6 units Expand High Fidelity PCR System (Roche). Cycling conditions were: 94 ◦ C for 4 min followed by 25 cycles of 94 ◦ C for 15 s; 50 ◦ C for 30 s, 72 ◦ C for 1 min, and then 1 cycle at 72 ◦ C for 7 min. The PCR product was cloned into pET-21a(+) for protein expression, and the insert was sequenced to ensure authenticity. Both human dPGM and O. volvulus iPGM were produced in T7 Express Competent Escherichia coli (New England Biolabs C2566H). Conditions were optimized to maximize expression, solubility and yield of each recombinant protein. The His-tagged proteins were extracted and purified on nickel resin (Qiagen) under native conditions according to the manufacturer’s instructions. An elution buffer (40 mM NaH2 PO4 , 300 mM NaCl, pH 8.0) containing varying amounts of imidazole was evaluated for optimal release of the His-tagged proteins from the nickel resin. Purity of the protein was estimated by 4–20% SDS-PAGE, and the protein concentration determined using the Bradford assay. For long-term storage, the protein was stored at −20 ◦ C in 50% glycerol. Prior to use, the enzymes were dialyzed against a phosphate buffer (40 mM NaH2 PO4 , 300 mM NaCl, pH 8.0, 5% glycerol) and stored for 1–2 months at 4 ◦ C. Recombinant iPGM enzymes from C. elegans (Ce-iPGM) [15], B. malayi (Bm-iPGM) [15] and T. brucei (Tb-iPGM) [17] were expressed and purified as described previously. 2.3. PGM enzyme assays Unless otherwise stated, PGM activity was measured using a standard one-step enzyme-coupled assay [1,18]. Activity was determined in the forward direction (3-PG to 2-PG) by measur-

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ing the consumption of NADH, which is monitored at 340 nm. The amount of NADH oxidized to NAD corresponds to the amount of enzyme product (2-PG) produced in the reaction. Reactions were performed at room temperature for 5 min with data collected at 10 s intervals using a Beckman DU 640 spectrophotometer. PGM (1.0 ␮g unless otherwise stated) was added to 1 ml assay buffer (30 mM Tris–HCl pH 7.0, 5 mM MgSO4 , 20 mM KCl), 1 mM ADP, 0.15 mM NADH, containing 10 mM 3-PG (Sigma), 2.5 units each of enolase (Sigma E6126), pyruvate kinase (Sigma P7768) and l-lactic dehydrogenase (Sigma L2518). Experiments were performed in duplicate and repeated at least twice. Control assays were also performed to provide a baseline (without using iPGM) and to ensure the function of the coupling enzymes (using 2-PG). One unit of PGM activity is defined as the conversion of 1.0 ␮mole NADH to NAD per minute. Mean apparent Km values were determined using 3-PG at concentrations ranging from 0.005 mM to 1.6 mM. Optimum curve fitting of data to the Michaelis–Menten equation was calculated using Microsoft Excel and Deltagraph programs. A two-step assay was performed to determine pH and temperature optima. During the first step of the assay in which the enzyme converts 3-PG to 2-PG the parameter under investigation was varied. The amount of enzyme used and time interval was optimized for each enzyme (O. volvulus 1.0 ␮g/2 min; B. malayi 0.25 ␮g/2 min; C. elegans 0.5 ␮g/2 min; human 0.25 ␮g/2.5 min) in order to measure the initial velocity of the reaction during which time there is no apparent product inhibition. The reaction was then stopped by boiling the sample for 5 min before dilution in an appropriate volume of standard assay buffer. In the second step, the amount of 2-PG produced was determined using the above standard assay. Conditions were optimized to measure sensitivity of the enzymes to vanadate, and to achieve inactivation of recombinant enzymes with EDTA followed by reactivation with divalent metal ions.

enzyme [7,19]. These residues are conserved in the other nematode enzymes and also in iPGMs from L. mexicana and T. brucei [13,14]. 3.2. Expression and purification of recombinant Ov-iPGM Recombinant Ov-iPGM was expressed in E. coli with a His-tag at the C-terminus and purified by nickel-affinity chromatography (Fig. S2). A number of experiments were performed to maximize expression, solubility and yield. These included varying growth temperature, timing and length of induction with varying amounts of isopropyl ␤-d-thiogalactopyranoside (IPTG). In addition, elution buffer containing different amounts of imidazole were evaluated for most effective release of His-tagged protein from the resin. Optimum conditions for production of soluble recombinant Ov-iPGM (16 mg/l of culture) involved growth of cultures at 37 ◦ C, induction with 0.1 mM IPTG for 3 h at 37 ◦ C, and use of an elution buffer containing 50 mM imidazole. The apparent molecular weight (58 kDa) on SDS-PAGE was consistent with the predicted molecular size (Fig. S2). 3.3. Biochemical characterization Recombinant Ov-iPGM was found to be a typical iPGM (Fig. 1). iPGM, unlike dPGM, does not require a cofactor and is not sensitive to vanadate. The addition of the cofactor, or preincubation with 100 ␮M sodium metavanadate for 15 min prior to performing the standard assay, was found to have no effect on the activity of Ov-iPGM (Fig. 1) or the activities of the other nematode enzymes, Bm-iPGM and Ce-iPGM (data not shown). In contrast, 94% of the activity of recombinant human dPGM was inhibited using the same concentration of vanadate (Fig. 1). Since pH has been shown to regulate the activity of iPGM from gram-positive, endospore forming bacteria [20–22], and

3. Results 3.1. Sequence analysis of Ov-iPGM The complete Ov-iPGM ORF (GenBankTM accession number AY640434) amplified from cDNA clone (EST SWOvL3CAN18E07) is 1548 bp in length. The translated protein (515 amino acids) has a predicted molecular mass of 57.2 kDa with a pI of 6.55. Sequence alignment of several iPGM enzymes (Fig. S1) indicates that Ov-iPGM is a close ortholog of the previously described Ce-iPGM and Bm-iPGM enzymes. OviPGM shares 93% and 72% sequence identity with Bm-iPGM and Ce-iPGM respectively, and is more closely related to the Bacillus stearothermophilus iPGM (42% identity) than to the kinetoplastid (T. brucei and L. mexicana) enzymes (28–29% identity). Ov-iPGM contains the highly conserved catalytic serine and 13 other residues (Fig. S1) predicted to be involved in catalysis based on structural and biochemical studies on B. stearothermophilus iPGM [7,19], including the residues which were shown to contact two metal ions (Fig. S1) in the bacterial

Fig. 1. Activity of recombinant O. volvulus iPGM and insensitivity to vanadate. Ov-iPGM with () and without () vanadate treatment; human dPGM with (䊉) and without () vanadate treatment. A control without iPGM (—) was included. Conversion of 3-PG to 2-PG is indicated by a decrease in NADH concentration as measured by its absorbance at 340 nM. The consumption of NADH is directly proportional to PGM activity.

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Fig. 2. Determination of pH and temperature optima for nematode iPGM and human dPGM. (A) Effect of pH on PGM activity. Enzymes and substrate (3-PG) were incubated at 25 ◦ C in buffers of differing pH values. The percentage relative activity at the various pH values tested is indicated for Ov-iPGM (), Bm-iPGM (*) and Ce-iPGM (). Human dPGM () was included for comparison. (B) Effect of temperature on PGM activity. Enzymes and substrate (3-PG) were incubated at various temperatures in buffer at pH 7. The percentage relative activity at the various temperatures tested is indicated for Ov-iPGM (), Bm-iPGM (*) and Ce-iPGM (). Human dPGM () was included for comparison.

the stability of bacterial and plant enzymes [20,23], the effect of pH on the activities of the nematode enzymes was determined. A two-step assay was performed in which the first step involved conversion of 10 mM 3-PG to 2-PG in either 30 mM Bis–Tris buffer at pH 6–7, or 30 mM Tris buffer at pH 7–9, each containing 5 mM MgSO4 and 20 mM KCl in a total volume of 200 ␮l. Following inactivation of the enzyme, a 50 ␮l aliquot was removed and the amount of 2-PG formed was measured using the coupled reaction. The nematode enzymes displayed maximum activity in the pH range 7.5–8.5 with considerable activity (at least 85%) at pH 9 (Fig. 2A). The lack of linearity around pH 7 may reflect the less efficient buffering capacity of the Tris and Bis–Tris buffers at this pH. Approximately 33–54% levels of activity were observed at the lowest pH tested (pH 6). Human dPGM also showed maximum activity around neutral pH. However this enzyme was more active in the lower pH range and less active at alkaline pH. The effect of temperature on activity was also determined using the two-step assay (Fig. 2B). The filarial and C. elegans enzymes showed high levels (>83%) of activity within the same temperature range of 17–32 ◦ C. Ce-iPGM was most active at 22 ◦ C, while the activities of the filarial enzymes peaked at 32 ◦ C. At temperatures greater than 37 ◦ C, the activities of all nematode enzymes decreased significantly. A substantially different activity profile was found for human dPGM. This enzyme was more active at higher temperatures with maximum activity at 52–57 ◦ C. To determine if nematode iPGMs require divalent ions, the enzymes (final concentration 0.5 mg/ml) were incubated with

various amounts of EDTA (0.01–6.25 mM) for 1 h at 30 ◦ C. Activities were then measured following dilution (1:1000) of the enzymes in standard assay buffer. Tb-iPGM was treated similarly to serve as a positive control. A concentration of 250 ␮M consistently resulted in an inactivation of nematode enzymes in the range of 46–83%. The nematode enzymes were particularly sensitive to EDTA since only 17% inhibition was obtained for Tb-iPGM treated similarly. Longer incubation times and higher temperatures resulted in complete and irreversible inactivation (data not shown). Following inactivation with 250 ␮M EDTA, the nematode enzymes (0.5 ␮g/ml) were incubated for 1 h at 37 ◦ C in standard assay buffer containing one of several divalent ions (100 ␮M magnesium, Mg2+ ; manganese, Mn2+ ; cobalt, Co2+ ; or nickel, Ni2+ ) for reactivation, and enzyme activity measured as described above. Reactivation of Tb-iPGM activity using cobalt ions was included as a positive control (data not shown) using the conditions previously described [13]. Similar reactivation profiles were obtained for each nematode enzyme (Fig. 3A–C). Substantial activity was restored by the addition of Mg2+ and Mn2+ ions. Cobalt and nickel ions were less potent. Calcium and iron were also evaluated and no activation was observed (data not shown). In these experiments the enzymes were partially inactivated since reactivation was unsuccessful once the enzymes were completely inactivated. Using optimum conditions for activity, several kinetic constants for the three nematode enzymes were determined using the Michaelis–Menten equation (Fig. 4 and Table 1). Similar apparent Km values were obtained. The catalytic efficiencies

Table 1 Kinetic activities of O. volvulus, C. elegans and B. malayi iPGM Protein

Specific activity (␮mol min−1 mg−1 )

Apparent Km for 3 PG (mM)

kcat (s−1 )

kcat /Km (M−1 s−1 )

Ov-iPGM Ce-iPGM Bm-iPGM

95 71 115

0.301 ± 0.01 0.508 ± 0.10 0.353 ± 0.02

83 ± 1.9 72 ± 5.9 110 ± 2.0

2.76E+05 1.42E+05 3.11E+05

Values are the average of at least three experiments ± S.D.

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Fig. 4. Measurement of the kinetic parameters of nematode iPGMs. The kinetic parameters were measured in the forward direction (glycolytic) for Ov-iPGM (), Bm-iPGM (*) and Ce-iPGM () using varying amounts of 3-PG (5 ␮M to 1.6 mM). The data were displayed using the Michaelis–Menten equation (v = F (S)). The error bar was determined from triplicate experiments.

(kcat /Km ) of the filarial enzymes were slightly higher than that of Ce-iPGM. The specific activities for the recombinant nematode enzymes were determined using optimum conditions for pH, temperature and substrate concentration (1.5 mM). Comparable values were obtained (Table 1), and are similar to those of Tb-iPGM and Hs-dPGM enzymes (data not shown). 4. Discussion

Fig. 3. Ion requirement and preference of nematode iPGM. Reactivation of the various nematode iPGM enzymes with divalent ions. EDTA-treated (250 ␮M) enzymes (0.5 ␮g/ml) were incubated for 1 h at 37 ◦ C in the presence of different ions namely, Mg2+ , Mn2+ , Co2+ , Ni2+ at a concentration of 100 ␮M and then assayed for activity. The activities of Ov-iPGM (Panel A), Bm-iPGM (Panel B) and Ce-iPGM (Panel C) were measured in a standard coupled assay. The dotted line indicates the level of activity after EDTA inactivation and prior to reactivation with the various divalent ions. One hundred percent activity is the level obtained for enzymes treated similarly (buffer, temperature, incubation time) but in the absence of EDTA. The results shown represent the mean (and S.D.) of at least three experiments.

The enzyme iPGM, responsible for the interconversion of 3PG and 2-PG in glycolysis and gluconeogenesis, has been the focus of considerable attention as a potential drug target in bacteria [7,8] and trypansosomes [14,17,24,25], since it plays a key role in development and is distinct from the evolutionarily unrelated enzyme dPGM, present in vertebrates. In T. brucei, RNAi studies have shown that iPGM is required for growth in procyclic [17] and trypomastigote [24] forms. iPGM has been proposed as a drug target in spore-forming Bacillus species [8,26]. Deletion of the gene in Bacillus subtilis resulted in extremely slow growth and an inability to form spores [27]. Similarly, in the non-spore forming bacterium Pseudomonas syringae, a tomato pathogen, iPGM was demonstrated to be essential for growth and infectivity [28]. Recent RNAi studies in the metazoan C. elegans have also revealed the importance of iPGM in nematodes, since down regulation of the transcript leads to embryonic and larval lethality [15]. The distribution of iPGM and dPGM in nature is considered to be haphazard and unpredictable. With the exception of vertebrates (dPGM) and higher plants (iPGM), related organisms may possess one or other form, or both [1,2]. In the phylum Nematoda, ESTs corresponding to only the iPGM form have been found among the ∼400,000 EST sequences from over 30 diverse parasitic nematode species that represent the var-

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ious major clades. iPGM was identified in 12 species that are responsible for diseases in plants, animals and humans [15]. This suggests that iPGM is universally expressed among nematodes and worthy of further investigation as a possible broad-spectrum nematode drug target. In this study we cloned and produced active iPGM from O. volvulus, an important human filarial parasite. We performed the first detailed characterization of the catalytic activities of three nematode iPGM enzymes. The results obtained provide further evidence for the presence of a conserved iPGM in nematodes. A specific iPGM activity was detected in lysates prepared from adult worms of the animal filarial parasite Dirofilaria immitis (data not shown). The filarial enzymes share a high degree of amino acid identity and are also related to C. elegans iPGM, suggesting that they may share similar catalytic properties. Like other iPGM enzymes, the activities of the nematode enzymes were found to be independent of cofactor and insensitive to vanadate. We evaluated the effect of pH on nematode iPGM activity since pH has been shown to modulate the activities of bacterial and trypanosomatid iPGM enzymes, and the stability of plant and bacterial enzymes [20,23]. We found all three nematode enzymes preferred neutral/mildly alkaline pH. The pH optima reported for the Leishmania [14] and bacterial iPGM enzymes [20,21] were also in this range. More detailed studies have shown that pH influences the three metal-binding histidine residues of Bacillus iPGM [26]. These results are consistent with iPGM enzymes belonging to the alkaline phosphatase superfamily [2]. In contrast, dPGM is a member of the acid phosphatase superfamily [2], and as shown here it is more active at neutral/mildly acid pH. Interestingly, the nematode enzymes displayed high levels of activity within a broad temperature range (17–32 ◦ C). This is perhaps not totally surprising since free-living nematodes and filarial parasites survive in environments with variable temperatures, either in the soil (C. elegans) or during development in insect and mammalian hosts (filarial parasites). Human dPGM was more active at higher temperatures with maximum activity at 52–57 ◦ C. Divalent metal ions are required for iPGM activity where they participate in the formation and dissociation of the phosphoserine intermediate [26]. A particularly interesting aspect of this interaction is the preference for a particular ion(s). Kinetoplastid iPGM activity has been shown to require cobalt [13,14], whereas manganese is preferred in the case of Bacillus species [10,11]. Sequence analysis indicated that the filarial and C. elegans iPGMs possess the 14 residues predicted to be involved in catalysis, including the catalytic serine [7,19]. Some of these residues are involved in binding two metal ions [7,19,29]. While the presence of the residues is suggestive of a metalloprotein, they do not offer any indication of ion specificity. The requirement for divalent metal ions for catalysis and/or structural integrity was indicated by loss of enzyme activity following incubation of the nematode iPGMs in the presence of EDTA. These enzymes were considerably more sensitive to EDTA than Tb-iPGM. The inactivation was reversible depending on the conditions used, with substantial levels of activity restored by the addition of magnesium or man-

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ganese ions. These findings are consistent with the nematode enzymes being more closely related to the B. stearothermophilus iPGM (manganese preference) than the kinetoplastid (cobalt preference) enzymes. The strong preference for cobalt among kinetoplastid iPGMs may relate to the possibility that trypanosomatids acquired many plant-like enzymes from an algal-like endosymbiont in an ancestral organism [30]. This is supported in the case of iPGM by the fact that higher plants possess an iPGM that functions in the presence of cobalt or manganese ions [31]. Interestingly, filarial parasites contain an obligate bacterial endosymbiont Wolbachia. Studies are underway to determine if this organism has an active PGM. Following optimization of conditions for maximum activity we determined levels of specific activity and catalytic efficiencies (kcat /Km ). We demonstrated that the iPGM enzymes from two important filarial parasites possess similar catalytic activities to the C. elegans iPGM enzyme. Therefore, despite an evolutionary distance of 350 million years since their last common ancestor, parasitic and free-living nematodes possess highly conserved iPGM enzymes, further substantiating the use of C. elegans as a model to understand the function of iPGM in parasitic species [15]. The kinetic activities obtained in this study are similar to those reported for the iPGMs from other organisms [1,13,14] suggesting the enzymes have similar efficiency despite sequence divergence. It was of importance to determine the optimal conditions for activity to facilitate the design of sensitive assays to screen for inhibitors. The completely distinct structures and catalytic mechanism of iPGM and dPGM enzymes offer great promise for the discovery of inhibitors with high selectivity for the nematode enzymes. Our results indicate that a single enzyme inhibitor would likely be effective against all nematode enzymes and mimic the RNAi phenotype observed in C. elegans. The identification of an inhibitor of iPGM would be highly significant as none exist, and could lead to the discovery of new therapies for a number of important parasitic and microbial diseases. Acknowledgements We gratefully acknowledge financial support from New England Biolabs and encouragement from Dr. Donald Comb. This work was also supported in part by a National Institute of Allergy and Infectious Diseases Small Business Innovation Research Grant AI061865. We thank Dr. Kshitiz Chaudhary for assistance in data analysis, Dr. Kshitiz Chaudhary and Dr. Jacopo Novelli for comments on the manuscript, and Scott Zimmer for assistance in cloning and expressing human dPGM. We also thank the Filarial Genome Project ([email protected]) for the O. volvulus EST clones and Dr. R. Prioli for the gift of the human cDNA. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molbiopara.2007.08.002.

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