- Email: [email protected]
Trypanosoma cruzi: molecular cloning and characterization of the S-adenosylhomocysteine hydrolase
Experimental Parasitology 105 (2003) 149–158 www.elsevier.com/locate/yexpr
Trypanosoma cruzi: molecular cloning and characterization of the S-adenosylhomocysteine hydrolaseq Nathan B. Parker,a Xiaoda Yang,b,1 Jens Hanke,c,2 Kenneth A. Mason,a,3 Richard L. Schowen,a,b Ronald T. Borchardt,b and Daniel H. Yinb,* a Department of Molecular Biosciences, The University of Kansas, Lawrence, KS 66045, USA Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66045, USA Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany b
c
Received 16 May 2003; received in revised form 29 September 2003; accepted 1 October 2003
Abstract S-Adenosylhomocysteine (AdoHcy) hydrolase has emerged as an attractive target for antiparasitic drug design because of its role in the regulation of all S-adenosylmethionine-dependent transmethylation reactions, including those reactions crucial for parasite replication. From a genomic DNA library of Trypanosoma cruzi, we have isolated a gene that encodes a polypeptide containing a highly conserved AdoHcy hydrolase consensus sequence. The recombinant T. cruzi enzyme was overexpressed in Escherichia coli and purified as a homotetramer. At pH 7.2 and 37 °C, the purified enzyme hydrolyzes AdoHcy to adenosine and homocysteine with a first-order rate constant of 1 s1 and synthesizes AdoHcy from adenosine and homocysteine with a pseudo-first-order rate constant of 3 s1 in the presence of 1 mM homocysteine. The reversible catalysis depends on the binding of NADþ to the enzyme. In spite of the significant structural homology between the parasitic and human AdoHcy hydrolase, the Kd of 1.3 lM for NADþ binding to the T. cruzi enzyme is approximately 11-fold higher than the Kd (0.12 lM) for NADþ binding to the human enzyme. Ó 2003 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: S-Adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1); NADþ -dependent enzyme; Trypanosoma cruzi (T. cruzi); Leishmania donovani (L. donovani); Plasmodium falciparum (P. falciparum); Ado, adenosine; AdoMet, S-adenosylmethionine; C3 NepA, 3-deazaneplanocin A; EST, expressed sequence tag; FPLC, fast protein liquid chromatography; Hcy, homocysteine; Ino, inosine; IPTG, isopropyl b-D -1-thiogalactopyranoside; PCR, polymerase chain reaction; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
1. Introduction Infection of the protozoan parasite Trypanosoma cruzi in humans causes ChagasÕ disease, a public health q The sequence data reported herein have been deposited in GenBank under Accession No. AY233397. * Corresponding author. Present address: Merck & Co., Inc., WP78-302, P.O. Box 4, West Point, PA 19486 USA. Fax: 1-215-6525299. E-mail addresses: [email protected] (J. Hanke), kamason @purdue.edu (K.A. Mason), [email protected] (D.H. Yin). 1 Present address: Department of Chemical Biology, School of Pharmaceutical Science, Peking University Health Science Center, Beijing 100083, China. 2 Present address: GPC Biotech AG, Fraunhoferstraße 20, 82152 Martinsried, Germany. 3 Present address: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
0014-4894/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2003.10.001
threat widely distributed from Central America to South America (Araujo-Jorge, 1999). The overall prevalence of human T. cruzi infection is estimated to be approximately 16–18 million cases with 90 million people living at risk (WHO, 1991). The lesions of ChagasÕ disease are incurable in the chronic stage, and current drug treatment is effective only for the initial infection and has substantial toxicity (Kirchhoff, 1993). Therefore, there is an acute need to exploit molecular targets unique to the parasite for the design of anti-trypanosoma drugs that are efficacious and safe. In recent years, S-adenosylhomocysteine (AdoHcy) hydrolase has emerged as a potential molecular target for the design of antiparasitic drugs (Henderson et al., 1992; Yang and Borchardt, 2000; Yin et al., 2000b). This enzyme is an attractive target in part because parasites express their own AdoHcy hydrolase with characteristics
150
N.B. Parker et al. / Experimental Parasitology 105 (2003) 149–158
(e.g., NADþ binding constants) that are different from those of the human enzyme (Yang and Borchardt, 2000). AdoHcy is a competitive product inhibitor of all S-adenosylmethionine (AdoMet)-dependent transmethylation reactions (Chiang, 1998; Turner et al., 2000). To sustain activity in transmethylation reactions in parasites, AdoHcy is reversibly hydrolyzed by AdoHcy hydrolase to adenosine (Ado) and homocysteine (Hcy). Interruption of this critical pathway of AdoHcy metabolism leads to accumulation of AdoHcy and inhibition of transmethylation reactions that are essential for the maturation of parasitic mRNA and biosynthesis of sterols as growth factors in parasites (Bacchi et al., 1995; Freistadt et al., 1988; Phelouzat et al., 1992; Urbina, 1999). For example, Henderson et al. (1992) have shown that AdoHcy hydrolase inhibitors suppress the growth of Leishmania donovani promastigotes, the parasite that causes leishmaniasis, and that this effect can be correlated with increases in the ratio of AdoHcy/AdoMet. Similar effects have been observed with Plasmodium falciparum, the parasite that causes malaria (Bitonti et al., 1990; Kitade et al., 1999; Whaun et al., 1986). Several competitive inhibitors of AdoHcy hydrolase, including 30 -deoxyadenosine, also markedly inhibited T. cruzi proliferation (Nakajima-Shimada et al., 1996). To date, the AdoHcy hydrolase genes from the parasites L. donovani, P. falciparum, and Trichomonas vaginalis have been cloned and the protein products have been overexpressed in Escherichia coli (Creedon et al., 1994; Henderson et al., 1992; Minotto et al., 1998; Yang and Borchardt, 2000). Despite the fact that these parasite enzymes have considerable amino acid sequence identity, interesting differences have been observed with respect to their binding of known inhibitors of AdoHcy hydrolase. For example, Leishmania and Trypanosoma parasites respond quite differently to 3-deazaneplanocin A (C3 NepA), a specific mechanism-based inhibitor of the human AdoHcy hydrolase (Avila et al., 1997). C3 NepA is a potent antileishmanial agent in vitro and in vivo, but is inactive against T. cruzi, suggesting that AdoHcy hydrolases produced by Leishmania and Trypanosoma exhibit different inhibitor specificities. Interesting differences in response to potential inhibitors have also been observed between the human AdoHcy hydrolase and a parasitic (L. donovani) form of the enzyme (Yang and Borchardt, 2000). For example, 30 -deoxyadenosine, a very poor competitive inhibitor of the human AdoHcy hydrolase, has been shown to produce time-dependent inactivation of the L. donovani form of the enzyme. The inhibitory effects of this nucleotide on the L. donovani enzyme appear to be related to its ability to bind to the NADþ binding site rather than the substrate binding site (X. Yang, R.T. Borchardt, R.L. Schowen, unpublished work). To determine whether the strategy of inhibiting the binding of NADþ to the parasitic AdoHcy hydrolase is
unique to L. donovani or general to parasite enzymes, we undertook the isolation of the AdoHcy hydrolase gene from T. cruzi and development of a viable expression system using E. coli to produce larger quantities of the enzyme for characterization of its structure and catalytic capabilities. In a survey of the active genes in T. cruzi performed by analyzing expressed sequence tags (ESTs) generated from a normalized epimastigote cDNA library, nucleotide sequences with similarity to portions of the L. donovani AdoHcy hydrolase gene were identified (Porcel et al., 2000). In this paper we report the isolation of a complete T. cruzi AdoHcy hydrolase gene by screening the high-density array of a T. cruzi genomic DNA library with probes generated using L. donovani AdoHcy hydrolase cDNA. In addition, we have demonstrated that the recombinant T. cruzi AdoHcy hydrolase purified from E. coli has specific NADþ -dependent AdoHcy hydrolase activity for either the hydrolysis of AdoHcy to Ado and Hcy or the synthesis of AdoHcy from Ado and Hcy.
2. Materials and methods 2.1. Screening of a T. cruzi genomic library for the complete AdoHcy hydrolase gene A cosmid library of 36864 primary clones with a mean insert size of 37 kb was constructed previously from total genomic DNA of a T. cruzi reference strain CL-Brener (Hanke et al., 1996). The library was arrayed in high-density on two nylon filters (22 22 cm). Each clone was spotted in duplicate to increase the redundancy and to facilitate the scoring of the hybridizations. Using a random priming kit (Megaprime, Amersham– Pharmacia, Piscataway, NJ), [a-32 P]dCTP labeled probes for screening the library were generated from a full-length L. donovani AdoHcy hydrolase cDNA (Henderson et al., 1992; Yang and Borchardt, 2000). Prior to hybridization, the filters were incubated in Church buffer (0.5 M sodium phosphate buffer, pH 7.2, containing 7% SDS, 1 mM EDTA, with addition of 0.1 mg/ml yeast tRNA) for 2 h at 65 °C (Sambrook et al., 1989). Hybridization to the filters was carried out using probes of 5 105 cpm/ml in 10 ml of Church buffer at 65 °C overnight. The hybridized filters were briefly rinsed twice at 65 °C in wash buffer (40 mM sodium phosphate buffer, pH 7.2, containing 0.1% SDS), then washed in 0.5–1 liter of wash buffer and slowly rocked in a water bath of 65 °C for 30 min. Subsequently, the filters were briefly blotted dry and film exposed overnight at –70 °C. The screening procedure yielded 14 positive clones. The cosmid clones were then grown in LB medium containing 30 g/ml kanamycin (Sambrook et al., 1989), and the cosmid DNA was prepared in accordance with the protocol of Qiagen Mini Plasmid Kit (Valencia,
N.B. Parker et al. / Experimental Parasitology 105 (2003) 149–158
151
CA) (Persson et al., 1998). For DNA sequencing, the cosmid DNA was transformed into E. coli strain XL1Blue (Stratagene, La Jolla, CA) and further purified using the Qiagen kit.
the Biotechnology Support Facility (The University of Kansas Medical Center, Kansas City, KS) using an ABI model 377 DNA Sequencer (Applied Biosystems, Foster City, CA).
2.2. Sequencing of the T. cruzi AdoHcy hydrolase gene
2.3. Construction of a recombinant T. cruzi AdoHcy hydrolase clone for protein expression
The cosmid DNA was initially sequenced with a primer, 50 -GGCTGCCTGCACATGAC-30 , based on a conserved nucleotide sequence about 150 bases downstream from the start codon of the AdoHcy hydrolase genes in L. donovani (Henderson et al., 1992), human (Coulter-Karis and Hershfield, 1989), rat (Merta et al., 1995), mouse (Bethin et al., 1995), and Drosophila melanogaster (Caggese et al., 1997), identified using the BLAST program with default search parameters (Altschul et al., 1997). Cosmid DNA from three of the fourteen clones yielded sequencing data, the rest clones may have incomplete sequences that can not hybridize to the probe. Subsequently, the sense and anti-sense primers were used in the sequencing to construct a fulllength T. cruzi AdoHcy hydrolase gene (Fig. 1). Oligonucleotides were synthesized by Sigma-Genosys (Woodlands, TX), and the sequencing was performed by
The polymerase chain reaction (PCR) was employed to amplify the open reading frame for the AdoHcy hydrolase from the cosmid DNA. Both the sense primer, 50 -GAATTCATGACCGATTACAAAGTCCG-30 with the Met initiation codon (boldface), and the anti-sense primer, 5-GAATTCTTAGTAGCGGTAGTGGTCC-30 with the termination codon (boldface), contained an EcoRI site (underlined). Amplification conditions were 95 °C for 1 min, 58 °C for 30 s, and 72 °C for 2 min for 40 cycles using Pfu Turbo polymerase (Stratagene) following the manufacturerÕs instructions. The amplified fragment was digested with restriction enzyme EcoRI (Promega, Madison, WI) and purified using QIAquick PCR Purification Kit (Qiagen). The purified fragment was ligated into a prokaryotic expression vector pPROK-1 containing promoter Ptac (Clontech, Palo
Fig. 1. Consensus nucleotide sequence of genomic DNA for T. cruzi AdoHcy hydrolase and deduced amino acid sequence of the enzyme. The DNA sequence is numbered from the first ATG codon. The initial methionine is assigned as number 1. Underlines and arrows show the positions of T. cruzi AdoHcy hydrolase gene for which primers were designed and the orientation in which they were sequenced.
152
N.B. Parker et al. / Experimental Parasitology 105 (2003) 149–158
Alto, CA) using T4 ligase (Promega) according to the protocols provided by the manufacturers (Yang and Borchardt, 2000). The recombinant plasmid was transformed into E. coli strain JM109. The cells were grown on LB-agar plates containing 75 lg/ml ampicillin (Sambrook et al., 1989). The clones containing recombinant plasmid with inserts in the right orientation were further selected by DNA sequencing using primers designed from positions on the vector upstream or downstream of the insert. 2.4. Overexpression and purification of T. cruzi AdoHcy hydrolase The transformed E. coli cells were grown in 2 YT medium containing 75 lg/ml ampicillin at 37 °C. Isopropyl-b-D -1-thiogalactopyranoside (IPTG) (1 mM) was added to induce the protein overexpression, and the temperature for cell growth was lowered to room temperature to avoid production of protein in an aggregated form. The cells were harvested after an additional 20 h of incubation. All of the subsequent protein purification procedures were carried out at 0–4 °C. The cell pellet was lysed in a Tris buffer (25 mM Tris, pH 7.2, containing 2 mM EDTA and 1 mg/ml lysozyme) followed by three freeze–thawing cycles. The cell-free lysate obtained from centrifugation was diluted with 1 volume of water and mixed with DEAE-cellulose at pH 7.2 (Sigma, St. Louis, MO). The protein was eluted from the DEAE-cellulose with a 100 mM potassium phosphate buffer (pH 7.2, containing 1 mM EDTA). The protein in the eluent was precipitated with 70% saturated (NH4 )2 SO4 . The collected pellet was dissolved buffer A (50 mM potassium phosphate buffer, pH 7.2, containing 1 mM EDTA) and applied to a size-exclusion gel filtration column (3 75 cm of Sephacryl S-300, Amersham–Pharmacia) pre-equilibrated with buffer A. Protein elution was monitored at 280 nm and fractions were analyzed for specific AdoHcy hydrolase activity (Henderson et al., 1992; Yang and Borchardt, 2000). The fractions containing AdoHcy hydrolase activity were pooled and further purified using a strong-anion exchange column (3 20 cm of Q-Sepharose, Amersham–Pharmacia) pre-equilibrated with buffer A. The fractions representing the first peak eluting from this weak-anion exchange column were collected, and (NH4 )2 SO4 precipitation followed by size-exclusion liquid chromatography as described above was used to obtain pure T. cruzi AdoHcy hydrolase. Protein concentrations were determined using Bradford reagents (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard (Sigma) (Bradford, 1976). The purity of the recombinant T. cruzi enzyme was assessed by SDS polyacrylamide gel electrophoresis. The T. cruzi apo AdoHcy hydrolase was prepared as previously described (Yuan et al., 1993).
2.5. Structural analysis of T. cruzi AdoHcy hydrolase The quaternary structure of the T. cruzi AdoHcy hydrolase was analyzed in 25 mM Tris buffer (pH 7.2) using a fast protein liquid chromatography (FPLC) system equipped with a size-exclusion column (Superdex 200 HR 10/30, Amersham–Pharmacia). The secondary structure of the enzyme was determined by circular dichroism (CD) spectroscopy. The CD spectra of 0.05 mg/ ml T. cruzi AdoHcy hydrolase in 25 mM Tris buffer (pH 7.2) were measured at 25 °C using a Jasco J-710 spectropolarimeter (Jasco, Tokyo, Japan) and a temperature-jacketed spectral cell with a pathlength of 1 cm. The apparent helical content was estimated using the program Contin, as previously described (Venyaminov and Yang, 1996; Yin et al., 2000a). 2.6. Assays of T. cruzi AdoHcy hydrolase activity Trypanosoma cruzi AdoHcy hydrolase activity was assayed in the synthetic direction by measuring the production rate of AdoHcy from Ado and Hcy using HPLC as previously described (Yuan et al., 1993). Quantitation of AdoHcy was accomplished using a UV detector set at 258 nm and a standard curve of AdoHcy (e258 ¼ 14; 900 M1 cm1 , Sigma) (Fasman, 1976). AdoHcy hydrolase activity in the hydrolytic direction was measured by monitoring the formation of the product Ado after its conversion to inosine using Ado deaminase. The conversion of Ado to inosine by Ado deaminase drives the AdoHcy hydrolase-catalyzed reaction in the hydrolytic direction (Spector, 1984; Turner et al., 2000). AdoHcy and inosine were separated on a C18 reversed-phase HPLC column (218TP54, Vydac, Hesperia, CA) equilibrated with 98% mobile phase A (10 mM 1-heptanesulfonic acid, 50 mM sodium phosphate, pH 3.2) and 2% mobile phase B (80% acetonitrile, 20% isopropanol). The elution gradient was 2–10% mobile phase B over 20 min at 1 ml/min flow rate. Inosine was monitored at 250 nm and quantified using a standard curve of inosine (e250 ¼ 12; 300 M1 cm1 , Sigma) (Fasman, 1976). 2.7. NADþ and NADH binding affinity The synthetic activity of T. cruzi AdoHcy hydrolase was measured using 1 lg/ml of the apo-enzyme, incubated with 1 mM Hcy and the indicated concentrations of NADþ in buffer A at 37 °C for 10 min. Ado (200 lM) was then added to start the reaction, and the samples were incubated at 37 °C for 10 min followed by addition of 50 lM HClO4 to quench the reaction. The data were fitted to a single-site binding model using the Origin Program (Microcal Software, Northampton, MA) as previously described to obtain the enzyme binding constant for NADþ , KdNAD (Yang and Borchardt, 2000).
N.B. Parker et al. / Experimental Parasitology 105 (2003) 149–158
Similarly, using 1 lg/ml apo-enzyme incubated with 100 lM NADþ and the indicated concentrations of NADH, the binding constant of KdNADH was calculated by fitting the data to the following equation: ½NADH KdNAD Activity ¼ Activitymax þ1 þ1 KdNADH ½NADþ
3. Results 3.1. Isolation and sequencing of the full T. cruzi AdoHcy hydrolase gene A survey of the active genes in T. cruzi performed by analyzing 5013 expressed sequence tags (ESTs) generated from a normalized epimastigote cDNA library has
153
shown homology between a segment of the T. cruzi cDNA and the L. donovani AdoHcy hydrolase gene (Porcel et al., 2000). The full-length L. donovani AdoHcy hydrolase cDNA was used as a template to generate 32 Plabeled PCR fragments by random priming. These fragments were used as probes for the isolation of the entire AdoHcy hydrolase gene locus from high density filters of an average 37 kb-insert genomic T. cruzi library (Hanke et al., 1996). On the basis of a genome size of the CL-Brener strain of around 45 Mb, the cosmid library would represent approximately 23 genome equivalents. Screening of the filters yielded 14 positive cosmid clones, three of which yielded sequencing data using a primer as described in Section 2. One of the positive clones, designated 62N9, was chosen for further analysis and was propagated and subjected to linear amplification sequencing. Our sequencing strategy produced the entire nucleotide sequence of the T. cruzi AdoHcy hydrolase
Fig. 2. AdoHcy hydrolase amino acid sequences alignment. Comparison of the deduced amino acid sequences of AdoHcy hydrolase from T. cruzi and other representative organisms was carried out using a program Lasergene from DNAStar (Madison, WI). GeneBank database Accession Nos. are M76556 (L. donovani); M61831 (human placental); NM017201 (rat). The dot ‘‘’’ indicates identical amino acid. ‘‘c’’ indicates residues involved in enzyme catalysis. ‘‘s’’ indicates residues involved in substrate binding. ‘‘n’’ indicates residues involved in cofactor NADþ binding. The solid line above and below the aligned sequences indicates significant non-conserved regions.
154
N.B. Parker et al. / Experimental Parasitology 105 (2003) 149–158
gene as well as several hundred nucleotides of 50 and 30 flanking sequences (Fig. 1). The entire nucleotide sequence was consolidated by sequencing of the complementary strand. The uninterrupted entire coding region of 1311 nt predicts a protein of 437 residues with a calculated molecular mass of 48,460 Da. The amino acid sequence corresponds to the proenzyme form of AdoHcy hydrolase, observed in all prokaryotic and eukaryotic species studied (Turner et al., 2000). Using the BLAST program at the website of the National Center for Biotechnology Information with default search parameters, the amino acid sequence of the T. cruzi AdoHcy hydrolase was compared to that of the enzyme from other organisms. The maximum gap alignment result shows that the T. cruzi enzyme is 70–73% identical to AdoHcy hydrolase from higher eukaryotes and 90–93% identical to AdoHcy hydrolase of other protozoa. For example, the overall sequence identity is 91 and 73% between the primary structure of the T. cruzi AdoHcy hydrolase and the corresponding enzymes from L. donovani and the human placenta, respectively (Fig. 2) (Coulter-Karis and Hershfield, 1989; Gomi et al., 1989; Henderson et al., 1992). The predicted amino acid sequence for the T. cruzi AdoHcy hydrolase is of the same length as that of L. donovani but five residues longer than that of mammalians. The sequence comparisons (Fig. 2) indicate that a greater similarity is seen between the T. cruzi and the L. donovani AdoHcy hydrolase than between the T. cruzi and the human or rat enzymes. The isoelectric points for the T. cruzi and the human AdoHcy hydrolase, as calculated on the basis of their amino acid sequences, differed significantly. However, pI values of 6.47 for the T. cruzi enzyme and 6.0 for the human enzyme show that both are acidic proteins. 3.2. Expression and purification of T. cruzi AdoHcy hydrolase The T. cruzi AdoHcy hydrolase gene was subcloned into an expression vector pPROK-1 at the EcoRI cloning site. The insertion was confirmed by restriction mapping with EcoRI. By automated DNA sequencing of the recombinant plasmid using primers from positions upstream and downstream of the insert, two clones with the insert in the right orientation were selected for protein overexpression. The T. cruzi AdoHcy hydrolase was expressed in the soluble form in the cell-free lysate as shown in Fig. 3 (lane 2). In the cell-free extract of E. coli, the recombinant enzyme represents approximately 30% of the total protein. After initial purification on a DEAE column, a crude product of about 70% purity was obtained (Fig. 3, lane 3). The purity was increased to 85% (Fig. 3, lane 4) after chromatography on a Sephacryl S-300 gel
Fig. 3. Coomassie blue-stained protein samples from each step of the purification. The recombinant T. cruzi AdoHcy hydrolase was purified as described in Section 2. The SDS acrylamide gel was loaded with protein molecular weight marker (lane 1); cell free extract of E. coli transfected with gene for T. cruzi AdoHcy hydrolase (lane 2); eluent from DEAE-cellulose (lane 3); fractions collected from size-exclusion chromatography (lane 4); fractions collected from ion-exchange chromatography (lane 5); and reconstituted and second size-exclusion column for purification of T. cruzi AdoHcy hydrolase (lane 6). The human recombinant AdoHcy hydrolase was used as a reference (lane 7).
filtration column. The next chromatography step using an anion-exchange column increased the purity to over 90%. The final step involving a second size-exclusion chromatography produced T. cruzi AdoHcy hydrolase of over 95% purity as shown by electrophoresis (Fig. 3, lane 6). About 20 mg of T. cruzi AdoHcy hydrolase can be routinely obtained from 1 liter of E. coli culture. In the presence of 100 lM NADþ at pH 7.2 and 37 °C, the recombinant T. cruzi enzyme catalyzes the interconversion of AdoHcy to adenosine (Ado) and homocysteine (Hcy) with a first-order rate constant of 1 s1 in the hydrolytic direction (AdoHcy ! Ado + Hcy) and a pseudo-first-order rate constant of 3 s1 in the synthetic direction (Ado + Hcy ! AdoHcy) in the presence of 1 mM Hcy. 3.3. Structure of T. cruzi AdoHcy hydrolase On SDS–PAGE, the recombinant T. cruzi enzyme migrated more slowly than the human AdoHcy hydrolase (Fig. 3, lane 7). The subunit molecular weight of the T. cruzi enzyme was estimated to be 48 3 kDa, which is similar to the predicted value (48.5 kDa) from the DNA sequence. Using size-exclusion semi-preparative FPLC, the T. cruzi enzyme elutes with an molecular weight of approximately 200 kDa, which is similar to that of human AdoHcy hydrolase (Fig. 4A). These results suggest that the T. cruzi enzyme exists as a tetramer similar to the human AdoHcy hydrolase. The secondary structure of the T. cruzi enzyme is nearly identical to that of the human AdoHcy hydrolase (Fig. 4B).
N.B. Parker et al. / Experimental Parasitology 105 (2003) 149–158
Fig. 4. Structural characterization of the purified T. cruzi AdoHcy hydrolase. Upper panel, chromatography of T. cruzi (solid line) and human AdoHcy hydrolase (dash-dot line) analyzed using a size-exclusion FPLC column as described in Section 2. Lower panel, CD spectra comparison of T. cruzi (solid circle) and human AdoHcy hydrolase (open circle).
3.4. Substrate kinetic properties of the recombinant T. cruzi AdoHcy hydrolase Fig. 5 shows that the activity of T. cruzi AdoHcy hydrolase is dependent on the concentration of the cofactor NADþ . When these data were fitted to a one-site binding model, a Kd of 1.3 0.1 lM was estimated for NADþ . Binding of NADH to T. cruzi AdoHcy hydrolase produced an inactive form of the enzyme. The Kd for NADH was estimated to be 104 7 nM from the data shown in Fig. 5 as described in Section 2. These data indicate that the affinity of T. cruzi AdoHcy hydrolase for NADH is about 13 times higher than that for NADþ .
4. Discussion Active AdoMet-dependent methyltransferases (e.g., guanine N 7 -methyltransferase, EC 2.1.1.33, and d24ð25Þ sterol methyltransferase, EC 2.1.1.41) have been identified in parasites, including Encephalitozoon cuniculi, L. donovani, Pneumocystis carinii, Trichostrongylus colubriformis, and T. cruzi (Frandsen and Bone, 1988; Hasne and Lawrence, 1999; Hausmann et al., 2002; Urbina et al., 1997; Urbina et al., 1995). Methylations of the
155
Fig. 5. The relationship between the enzyme activity and the concentration of NADþ and NADH in the reaction solution. The activities of T. cruzi AdoHcy hydrolase reconstituted in the apo-form were assayed in the synthetic direction at different concentrations of NADþ and NADH. Curves are results from the model fitting described in Section 2.
type catalyzed by guanine N 7 -methyltransferase and d24ð25Þ -sterol methyltransferase have been inferred to occur in T. cruzi from the presence of 50 -end methylated SL RNA and methylated sterols (Ullu and Tschudi, 1995; Urbina et al., 1996). Metabolism of AdoHcy, the product inhibitor of AdoMet-dependent methyltransferases, has been shown to be catalyzed by AdoHcy hydrolase produced in the parasites L. donovani, P. falciparum, and T. vaginalis (Creedon et al., 1994; Henderson et al., 1992; Minotto et al., 1998). As yet, AdoHcy hydrolase activity in T. cruzi has not been reported. However, sequencing data generated from a T. cruzi epimastigote normalized cDNA library, as part of the T. cruzi genome project, have presented seven clones with a cDNA sequence similar to part of the L. donovani AdoHcy hydrolase gene (Porcel et al., 2000). Using a computer alignment program (BLAST 2.0, Washington University, St. Louis, MO) to compare the T. cruzi AdoHcy hydrolase gene from our study to the seven clones in the cDNA library, we found that all the cDNA sequences (ranging from 237 to 549 bp) were covered by the full-length AdoHcy hydrolase gene from the genomic library of T. cruzi DNA with 96–100% identities to
156
N.B. Parker et al. / Experimental Parasitology 105 (2003) 149–158
corresponding regions (Korf and Gish, 2000). This comparison indicates that the T. cruzi AdoHcy hydrolase gene is expressed in this parasite. The amino acid sequences of AdoHcy hydrolase from mammalians and parasites have a high level of conservation during evolution. For example, the T. cruzi AdoHcy hydrolase sequence exhibits a high positive similarity score with the L. donovani sequence (93%) and with human and rat sequences (81%) by the Gapped BLAST program (Altschul et al., 1997). X-ray crystal structures of the human and rat AdoHcy hydrolase suggest certain amino acid residues to be involved in substrate and cofactor binding and in catalysis (Turner et al., 2000). The critical roles of these residues have also been confirmed by site-directed mutagenesis (unpublished data) and computational modeling (Hu et al., 2001). As shown in Fig. 2, most of these residues are conserved among the AdoHcy hydrolase sequences from T. cruzi, L. donovani, human, and rat. The one exception is residue 56, which is a Gln in the T. cruzi and L. donovani enzymes and Glu in the human and rat enzymes. With these highly conserved sequences, it is not surprising that the T. cruzi AdoHcy hydrolase has quaternary and secondary structures similar to those of the human enzyme (Fig. 4). However, the variable regions in the sequences apparently produce some differences in the properties of the T. cruzi and human AdoHcy hydrolase. One difference is the charge characteristics of the proteins. For example, human AdoHcy hydrolase has a calculated pI of 6.3, and it binds to a weak anion exchange column in 50 mM phosphate buffer at pH 7.2. In contrast, under the same conditions T. cruzi AdoHcy hydrolase, with a predicted pI of 6.5, does not bind to this weak anion exchange column. It is possible that the difference in their experimental pI values in solution may be significantly larger than their calculated pI values. If potent inhibitors of a parasite AdoHcy hydrolase (e.g., T. cruzi) with minimal effects on the human enzyme could be designed, these types of compounds might have clinical potential as antiparasitic agents. A difference in the properties of the parasite and human AdoHcy hydrolase that might be exploited for the drug design purpose is the binding of the cofactor NADþ . It is well known that mammalian AdoHcy hydrolase binds tightly one molecule of the cofactor per enzyme subunit (Palmer and Abeles, 1979). The enzyme is positively cooperative in binding NADþ (Gomi et al., 1989). The Kd value of human AdoHcy hydrolase is 120 20 nM for both NADþ and NADH (Yang and Borchardt, 2000). Release of the cofactors from the human enzyme is not normally observed due to the very slow dissociation rates (Gomi et al., 1989). In contrast, the T. cruzi enzyme binds NADþ (Kd ¼ 1:3 lM) more loosely to the four sites of the tetrameric enzyme. Similar results have been reported for the L. donovani enzyme (Yang and
Borchardt, 2000). However, the T. cruzi AdoHcy hydrolase has an affinity (Kd ¼ 104 nM) for NADH that is similar to that of the human enzyme but 20 times lower than the Kd for the L. donovani enzyme (Yang and Borchardt, 2000). From the sequence comparison, we observed three non-conserved regions close to the cofactor binding sites (Fig. 2). These regions may modulate the affinity of AdoHcy hydrolase for the cofactor. Further side-directed mutagenesis in these regions is needed to test our hypothesis. The differences found in the binding properties of T. cruzi AdoHcy hydrolase relative to those of the human enzyme for NADþ are being exploited in our laboratory for the design of antitrypanosoma drugs that selectively inhibit T. cruzi AdoHcy hydrolase.
Acknowledgments This work was supported in part by a grant from the National Institutes of Health (GM29332) and a Postdoctoral Fellowship to D.H.Y. from the American Heart Association Heartland Affiliate (9920522Z). We thank Dr. Jinsong Zhang, Dr. Yongbo Hu, and Ms. Mengmeng Wang, The University of Kansas, and Dr. P. Lynne Howell, Hospital for Sick Children, Toronto, Canada, for insightful discussions. We thank Clark Bloomer, Biotechnology Support Facility at University of Kansas Medical Center, for his work on DNA sequencing, and Nancy Harmony for proofreading and editing the manuscript.
References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389–3402. Araujo-Jorge, T.C., 1999. Biology and ultra-structure of Trypanosoma cruzi: a 90-year old challenge for scientists. Memorias do Instituto Oswaldo Cruz 94, 131–134. Avila, J.L., Avila, A., Polegre, M.A., Marquez, V.E., 1997. Specific inhibitory effect of 3-deazaneplanocin A against several Leishmania mexicana and L. braziliensis strains. American Journal of Tropical Medicine and Hygiene 57, 407–412. Bacchi, C.J., Goldberg, B., Garofalo-Hannan, J., Rattendi, D., Lyte, P., Yarlett, N., 1995. Fate of soluble methionine in African trypanosomes: effects of metabolic inhibitors. The Biochemical Journal 309, 737–743. Bethin, K.E., Cimato, T.R., Ettinger, M.J., 1995. Copper binding to mouse liver S-adenosylhomocysteine hydrolase and the effects of copper on its levels. Journal of Biological Chemistry 270, 20703– 20711. Bitonti, A.J., Baumann, R.J., Jarvi, E.T., McCarthy, J.R., McCann, P.P., 1990. Antimalarial activity of a 4Õ,5Õ-unsaturated 5Õ-fluoroadenosine mechanism-based inhibitor of S-adenosyl-L -homocysteine hydrolase. Biochemical Pharmacology 40, 601–606. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the
N.B. Parker et al. / Experimental Parasitology 105 (2003) 149–158 principle of protein–dye binding. Analytical Biochemistry 72, 248–254. Caggese, C., Ragone, G., Barsanti, P., Moschetti, R., Messina, A., Massari, S., Caizzi, R., 1997. The S-adenosyl-L -homocysteine hydrolase of Drosophila melanogaster: identification, deduced amino acid sequence and cytological localization of the structural gene. Molecular and General Genetics 253, 492–498. Chiang, P.K., 1998. Biological effects of inhibitors of S-adenosylhomocysteine hydrolase. Pharmacology and Therapeutics 77, 115– 134. Coulter-Karis, D.E., Hershfield, M.S., 1989. Sequence of full length cDNA for human S-adenosylhomocysteine hydrolase. Annals of Human Genetics 53, 169–175. Creedon, K.A., Rathod, P.K., Wellems, T.E., 1994. Plasmodium falciparum S -adenosylhomocysteine hydrolase. cDNA identification, predicted protein sequence, and expression in Escherichia coli. Journal of Biological Chemistry 269, 16364–16370. Fasman, G.D., 1976. In: Fasman, G.D. (Ed.), Handbook of Biochemistry and Molecular Biology: Nucleic Acid II. CRC Press, Cleveland, OH, pp. 97–120. Frandsen, J.C., Bone, L.W., 1988. Derivatives of epinephrine, norepinephrine, octopamine and histamine formed by homogenates of Trichostrongylus colubriformis, a nematode parasite of ruminants. Comparative Biochemistry and Physiology C 91, 385–387. Freistadt, M.S., Cross, G.A., Robertson, H.D., 1988. Discontinuously synthesized mRNA from Trypanosoma brucei contains the highly methylated 5Õ cap structure, m7GpppA*A*C(2Õ-O)mU*A. Journal of Biological Chemistry 263, 15071–15075. Gomi, T., Takata, Y., Fujioka, M., 1989. Rat liver S-adenosylhomocysteinase. Spectrophotometric study of coenzyme binding. Biochimica et Biophysica Acta 994, 172–179. Hanke, J., Sanchez, D.O., Henriksson, J., Aslund, L., Pettersson, U., Frasch, A.C., Hoheisel, J.D., 1996. Mapping the Trypanosoma cruzi genome: analyses of representative cosmid libraries. Biotechniques 21, 686–688, 690-693. Hasne, M.P., Lawrence, F., 1999. Characterization of prenylated protein methyltransferase in Leishmania. The Biochemical Journal 342 (Part 3), 513–518. Hausmann, S., Vivares, C.P., Shuman, S., 2002. Characterization of the mRNA capping apparatus of the microsporidian parasite Encephalitozoon cuniculi. Journal of Biological Chemistry 277, 96– 103. Henderson, D.M., Hanson, S., Allen, T., Wilson, K., Coulter-Karis, D.E., Greenberg, M.L., Hershfield, M.S., Ullman, B., 1992. Cloning of the gene encoding Leishmania donovani S -adenosylhomocysteine hydrolase, a potential target for antiparasitic chemotherapy. Molecular and Biochemical Parasitology 53, 169–183. Hu, Y., Yang, X., Yin, D.H., Mahadevan, J., Kuczera, K., Schowen, R.L., Borchardt, R.T., 2001. Computational characterization of substrate binding and catalysis in S-adenosylhomocysteine hydrolase. Biochemistry 40, 15143–15152. Kirchhoff, L.V., 1993. American trypanosomiasis (ChagasÕ disease)—a tropical disease now in the United States. The New England Journal of Medicine 329, 639–644. Kitade, Y., Kozaki, A., Gotoh, T., Miwa, T., Nakanishi, M., Yatome, C., 1999. Synthesis of S-adenosyl-L -homocysteine hydrolase inhibitors and their biological activities. Nucleic Acids Symposium Series 42, 25–26. Korf, I., Gish, W., 2000. MPBLAST: improved BLAST performance with multiplexed queries. Bioinformatics 16, 1052–1053. Merta, A., Aksamit, R.R., Kasir, J., Cantoni, G.L., 1995. The gene and pseudogenes of rat S-adenosyl-L -homocysteine hydrolase. European Journal of Biochemistry 229, 575–582. Minotto, L., Ko, G.A., Edwards, M.R., Bagnara, A.S., 1998. Trichomonas vaginalis: expression and characterisation of recombinant S-adenosylhomocysteinase. Experimental Parasitology 90, 175–180.
157
Nakajima-Shimada, J., Hirota, Y., Aoki, T., 1996. Inhibition of Trypanosoma cruzi growth in mammalian cells by purine and pyrimidine analogs. Antimicrobial Agents and Chemotherapy 40, 2455–2458. Palmer, J.L., Abeles, R.H., 1979. The mechanism of action of Sadenosylhomocysteinase. Journal of Biological Chemistry 254, 1217–1226. Persson, K., Aslund, L., Grahn, B., Hanke, J., Heby, O., 1998. Trypanosoma cruzi has not lost its S-adenosylmethionine decarboxylase: characterization of the gene and the encoded enzyme. Biochemical Journal 333, 527–537. Phelouzat, M.A., Lawrence, F., Moulay, L., Borot, C., Schaeverbeke, J., Schaeverbeke, M., Robert-Gero, M., 1992. Leishmania donovani: antagonistic effect of S-adenosyl methionine on ultrastructural changes and growth inhibition induced by sinefungin. Experimental Parasitology 74, 177–187. Porcel, B.M., Tran, A.N., Tammi, M., Nyarady, Z., Rydaker, M., Urmenyi, T.P., Rondinelli, E., Pettersson, U., Andersson, B., Aslund, L., 2000. Gene survey of the pathogenic protozoan Trypanosoma cruzi. Genome Research 10, 1103–1107. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Spector, T., 1984. Progress curve analysis of adenosine deaminasecatalyzed reactions. Analytical Biochemistry 138, 242–245. Turner, M.A., Yang, X., Yin, D., Kuczera, K., Borchardt, R.T., Howell, P.L., 2000. Structure and function of S-adenosylhomocysteine hydrolase. Cell Biochemistry and Biophysics 33, 101–125. Ullu, E., Tschudi, C., 1995. Accurate modification of the trypanosome spliced leader cap structure in a homologous cell-free system. Journal of Biological Chemistry 270, 20365–20369. Urbina, J.A., 1999. Parasitological cure of Chagas disease: is it possible? Is it relevant? Memorias do Instituto Oswaldo Cruz 94, 349–355. Urbina, J.A., Visbal, G., Contreras, L.M., McLaughlin, G., Docampo, R., 1997. Inhibitors of delta24(25) sterol methyltransferase block sterol synthesis and cell proliferation in Pneumocystis carinii. Antimicrobial Agents and Chemotherapy 41, 1428–1432. Urbina, J.A., Vivas, J., Lazardi, K., Molina, J., Payares, G., Piras, M.M., Piras, R., 1996. Antiproliferative effects of delta 24(25) sterol methyl transferase inhibitors on Trypanosoma (Schizotrypanum) cruzi: in vitro and in vivo studies. Chemotherapy 42, 294– 307. Urbina, J.A., Vivas, J., Visbal, G., Contreras, L.M., 1995. Modification of the sterol composition of Trypanosoma (Schizotrypanum) cruzi epimastigotes by delta 24(25)-sterol methyl transferase inhibitors and their combinations with ketoconazole. Molecular and Biochemical Parasitology 73, 199–210. Venyaminov, S.Y., Yang, J.T., 1996. Circular Dichroism and the Conformational Analysis of Biomolecules. Plenum Press, New York, NY. pp. 69–107. Whaun, J.M., Miura, G.A., Brown, N.D., Gordon, R.K., Chiang, P.K., 1986. Antimalarial activity of neplanocin A with perturbations in the metabolism of purines, polyamines and S-adenosylmethionine. The Journal of Pharmacology and Experimental Therapeutics 236, 277–283. WHO, 1991. Control of Chagas disease. Report of a WHO Expert Committee. World Health Organization Technical Report Series 811, pp. 1–95. Yang, X., Borchardt, R.T., 2000. Overexpression, purification, and characterization of S-adenosylhomocysteine hydrolase from Leishmania donovani. Archives of Biochemistry and Biophysics 383, 272– 280. Yin, D., Sun, H., Ferrington, D.A., Squier, T.C., 2000a. Closer proximity between opposing domains of vertebrate calmodulin following deletion of Met(145)–Lys(148). Biochemistry 39, 10255– 10268.
158
N.B. Parker et al. / Experimental Parasitology 105 (2003) 149–158
Yin, D., Yang, X., Borchardt, R.T., Yuan, C.-S., 2000b. Mechanismbased S-adenosyl-L -homocysteine hydrolase inhibitors in the search for broad-spectrum antiviral agents. In: Torrence, P.F. (Ed.), Biomedical Chemistry Applying Chemical Principles to the Understanding and Treatment of Disease. Wiley, New York, NY, pp. 41–71.
Yuan, C.S., Yeh, J., Squier, T.C., Rawitch, A., Borchardt, R.T., 1993. Ligand-dependent changes in intrinsic fluorescence of Sadenosylhomocysteine hydrolase: implications for the mechanism of inhibitor-induced inhibition. Biochemistry 32, 10414– 10422.
Trypanosoma cruzi: molecular cloning and characterization of the S-adenosylhomocysteine hydrolaseq Nathan B. Parker,a Xiaoda Yang,b,1 Jens Hanke,c,2 Kenneth A. Mason,a,3 Richard L. Schowen,a,b Ronald T. Borchardt,b and Daniel H. Yinb,* a Department of Molecular Biosciences, The University of Kansas, Lawrence, KS 66045, USA Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66045, USA Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany b
c
Received 16 May 2003; received in revised form 29 September 2003; accepted 1 October 2003
Abstract S-Adenosylhomocysteine (AdoHcy) hydrolase has emerged as an attractive target for antiparasitic drug design because of its role in the regulation of all S-adenosylmethionine-dependent transmethylation reactions, including those reactions crucial for parasite replication. From a genomic DNA library of Trypanosoma cruzi, we have isolated a gene that encodes a polypeptide containing a highly conserved AdoHcy hydrolase consensus sequence. The recombinant T. cruzi enzyme was overexpressed in Escherichia coli and purified as a homotetramer. At pH 7.2 and 37 °C, the purified enzyme hydrolyzes AdoHcy to adenosine and homocysteine with a first-order rate constant of 1 s1 and synthesizes AdoHcy from adenosine and homocysteine with a pseudo-first-order rate constant of 3 s1 in the presence of 1 mM homocysteine. The reversible catalysis depends on the binding of NADþ to the enzyme. In spite of the significant structural homology between the parasitic and human AdoHcy hydrolase, the Kd of 1.3 lM for NADþ binding to the T. cruzi enzyme is approximately 11-fold higher than the Kd (0.12 lM) for NADþ binding to the human enzyme. Ó 2003 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: S-Adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1); NADþ -dependent enzyme; Trypanosoma cruzi (T. cruzi); Leishmania donovani (L. donovani); Plasmodium falciparum (P. falciparum); Ado, adenosine; AdoMet, S-adenosylmethionine; C3 NepA, 3-deazaneplanocin A; EST, expressed sequence tag; FPLC, fast protein liquid chromatography; Hcy, homocysteine; Ino, inosine; IPTG, isopropyl b-D -1-thiogalactopyranoside; PCR, polymerase chain reaction; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
1. Introduction Infection of the protozoan parasite Trypanosoma cruzi in humans causes ChagasÕ disease, a public health q The sequence data reported herein have been deposited in GenBank under Accession No. AY233397. * Corresponding author. Present address: Merck & Co., Inc., WP78-302, P.O. Box 4, West Point, PA 19486 USA. Fax: 1-215-6525299. E-mail addresses: [email protected] (J. Hanke), kamason @purdue.edu (K.A. Mason), [email protected] (D.H. Yin). 1 Present address: Department of Chemical Biology, School of Pharmaceutical Science, Peking University Health Science Center, Beijing 100083, China. 2 Present address: GPC Biotech AG, Fraunhoferstraße 20, 82152 Martinsried, Germany. 3 Present address: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
0014-4894/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2003.10.001
threat widely distributed from Central America to South America (Araujo-Jorge, 1999). The overall prevalence of human T. cruzi infection is estimated to be approximately 16–18 million cases with 90 million people living at risk (WHO, 1991). The lesions of ChagasÕ disease are incurable in the chronic stage, and current drug treatment is effective only for the initial infection and has substantial toxicity (Kirchhoff, 1993). Therefore, there is an acute need to exploit molecular targets unique to the parasite for the design of anti-trypanosoma drugs that are efficacious and safe. In recent years, S-adenosylhomocysteine (AdoHcy) hydrolase has emerged as a potential molecular target for the design of antiparasitic drugs (Henderson et al., 1992; Yang and Borchardt, 2000; Yin et al., 2000b). This enzyme is an attractive target in part because parasites express their own AdoHcy hydrolase with characteristics
150
N.B. Parker et al. / Experimental Parasitology 105 (2003) 149–158
(e.g., NADþ binding constants) that are different from those of the human enzyme (Yang and Borchardt, 2000). AdoHcy is a competitive product inhibitor of all S-adenosylmethionine (AdoMet)-dependent transmethylation reactions (Chiang, 1998; Turner et al., 2000). To sustain activity in transmethylation reactions in parasites, AdoHcy is reversibly hydrolyzed by AdoHcy hydrolase to adenosine (Ado) and homocysteine (Hcy). Interruption of this critical pathway of AdoHcy metabolism leads to accumulation of AdoHcy and inhibition of transmethylation reactions that are essential for the maturation of parasitic mRNA and biosynthesis of sterols as growth factors in parasites (Bacchi et al., 1995; Freistadt et al., 1988; Phelouzat et al., 1992; Urbina, 1999). For example, Henderson et al. (1992) have shown that AdoHcy hydrolase inhibitors suppress the growth of Leishmania donovani promastigotes, the parasite that causes leishmaniasis, and that this effect can be correlated with increases in the ratio of AdoHcy/AdoMet. Similar effects have been observed with Plasmodium falciparum, the parasite that causes malaria (Bitonti et al., 1990; Kitade et al., 1999; Whaun et al., 1986). Several competitive inhibitors of AdoHcy hydrolase, including 30 -deoxyadenosine, also markedly inhibited T. cruzi proliferation (Nakajima-Shimada et al., 1996). To date, the AdoHcy hydrolase genes from the parasites L. donovani, P. falciparum, and Trichomonas vaginalis have been cloned and the protein products have been overexpressed in Escherichia coli (Creedon et al., 1994; Henderson et al., 1992; Minotto et al., 1998; Yang and Borchardt, 2000). Despite the fact that these parasite enzymes have considerable amino acid sequence identity, interesting differences have been observed with respect to their binding of known inhibitors of AdoHcy hydrolase. For example, Leishmania and Trypanosoma parasites respond quite differently to 3-deazaneplanocin A (C3 NepA), a specific mechanism-based inhibitor of the human AdoHcy hydrolase (Avila et al., 1997). C3 NepA is a potent antileishmanial agent in vitro and in vivo, but is inactive against T. cruzi, suggesting that AdoHcy hydrolases produced by Leishmania and Trypanosoma exhibit different inhibitor specificities. Interesting differences in response to potential inhibitors have also been observed between the human AdoHcy hydrolase and a parasitic (L. donovani) form of the enzyme (Yang and Borchardt, 2000). For example, 30 -deoxyadenosine, a very poor competitive inhibitor of the human AdoHcy hydrolase, has been shown to produce time-dependent inactivation of the L. donovani form of the enzyme. The inhibitory effects of this nucleotide on the L. donovani enzyme appear to be related to its ability to bind to the NADþ binding site rather than the substrate binding site (X. Yang, R.T. Borchardt, R.L. Schowen, unpublished work). To determine whether the strategy of inhibiting the binding of NADþ to the parasitic AdoHcy hydrolase is
unique to L. donovani or general to parasite enzymes, we undertook the isolation of the AdoHcy hydrolase gene from T. cruzi and development of a viable expression system using E. coli to produce larger quantities of the enzyme for characterization of its structure and catalytic capabilities. In a survey of the active genes in T. cruzi performed by analyzing expressed sequence tags (ESTs) generated from a normalized epimastigote cDNA library, nucleotide sequences with similarity to portions of the L. donovani AdoHcy hydrolase gene were identified (Porcel et al., 2000). In this paper we report the isolation of a complete T. cruzi AdoHcy hydrolase gene by screening the high-density array of a T. cruzi genomic DNA library with probes generated using L. donovani AdoHcy hydrolase cDNA. In addition, we have demonstrated that the recombinant T. cruzi AdoHcy hydrolase purified from E. coli has specific NADþ -dependent AdoHcy hydrolase activity for either the hydrolysis of AdoHcy to Ado and Hcy or the synthesis of AdoHcy from Ado and Hcy.
2. Materials and methods 2.1. Screening of a T. cruzi genomic library for the complete AdoHcy hydrolase gene A cosmid library of 36864 primary clones with a mean insert size of 37 kb was constructed previously from total genomic DNA of a T. cruzi reference strain CL-Brener (Hanke et al., 1996). The library was arrayed in high-density on two nylon filters (22 22 cm). Each clone was spotted in duplicate to increase the redundancy and to facilitate the scoring of the hybridizations. Using a random priming kit (Megaprime, Amersham– Pharmacia, Piscataway, NJ), [a-32 P]dCTP labeled probes for screening the library were generated from a full-length L. donovani AdoHcy hydrolase cDNA (Henderson et al., 1992; Yang and Borchardt, 2000). Prior to hybridization, the filters were incubated in Church buffer (0.5 M sodium phosphate buffer, pH 7.2, containing 7% SDS, 1 mM EDTA, with addition of 0.1 mg/ml yeast tRNA) for 2 h at 65 °C (Sambrook et al., 1989). Hybridization to the filters was carried out using probes of 5 105 cpm/ml in 10 ml of Church buffer at 65 °C overnight. The hybridized filters were briefly rinsed twice at 65 °C in wash buffer (40 mM sodium phosphate buffer, pH 7.2, containing 0.1% SDS), then washed in 0.5–1 liter of wash buffer and slowly rocked in a water bath of 65 °C for 30 min. Subsequently, the filters were briefly blotted dry and film exposed overnight at –70 °C. The screening procedure yielded 14 positive clones. The cosmid clones were then grown in LB medium containing 30 g/ml kanamycin (Sambrook et al., 1989), and the cosmid DNA was prepared in accordance with the protocol of Qiagen Mini Plasmid Kit (Valencia,
N.B. Parker et al. / Experimental Parasitology 105 (2003) 149–158
151
CA) (Persson et al., 1998). For DNA sequencing, the cosmid DNA was transformed into E. coli strain XL1Blue (Stratagene, La Jolla, CA) and further purified using the Qiagen kit.
the Biotechnology Support Facility (The University of Kansas Medical Center, Kansas City, KS) using an ABI model 377 DNA Sequencer (Applied Biosystems, Foster City, CA).
2.2. Sequencing of the T. cruzi AdoHcy hydrolase gene
2.3. Construction of a recombinant T. cruzi AdoHcy hydrolase clone for protein expression
The cosmid DNA was initially sequenced with a primer, 50 -GGCTGCCTGCACATGAC-30 , based on a conserved nucleotide sequence about 150 bases downstream from the start codon of the AdoHcy hydrolase genes in L. donovani (Henderson et al., 1992), human (Coulter-Karis and Hershfield, 1989), rat (Merta et al., 1995), mouse (Bethin et al., 1995), and Drosophila melanogaster (Caggese et al., 1997), identified using the BLAST program with default search parameters (Altschul et al., 1997). Cosmid DNA from three of the fourteen clones yielded sequencing data, the rest clones may have incomplete sequences that can not hybridize to the probe. Subsequently, the sense and anti-sense primers were used in the sequencing to construct a fulllength T. cruzi AdoHcy hydrolase gene (Fig. 1). Oligonucleotides were synthesized by Sigma-Genosys (Woodlands, TX), and the sequencing was performed by
The polymerase chain reaction (PCR) was employed to amplify the open reading frame for the AdoHcy hydrolase from the cosmid DNA. Both the sense primer, 50 -GAATTCATGACCGATTACAAAGTCCG-30 with the Met initiation codon (boldface), and the anti-sense primer, 5-GAATTCTTAGTAGCGGTAGTGGTCC-30 with the termination codon (boldface), contained an EcoRI site (underlined). Amplification conditions were 95 °C for 1 min, 58 °C for 30 s, and 72 °C for 2 min for 40 cycles using Pfu Turbo polymerase (Stratagene) following the manufacturerÕs instructions. The amplified fragment was digested with restriction enzyme EcoRI (Promega, Madison, WI) and purified using QIAquick PCR Purification Kit (Qiagen). The purified fragment was ligated into a prokaryotic expression vector pPROK-1 containing promoter Ptac (Clontech, Palo
Fig. 1. Consensus nucleotide sequence of genomic DNA for T. cruzi AdoHcy hydrolase and deduced amino acid sequence of the enzyme. The DNA sequence is numbered from the first ATG codon. The initial methionine is assigned as number 1. Underlines and arrows show the positions of T. cruzi AdoHcy hydrolase gene for which primers were designed and the orientation in which they were sequenced.
152
N.B. Parker et al. / Experimental Parasitology 105 (2003) 149–158
Alto, CA) using T4 ligase (Promega) according to the protocols provided by the manufacturers (Yang and Borchardt, 2000). The recombinant plasmid was transformed into E. coli strain JM109. The cells were grown on LB-agar plates containing 75 lg/ml ampicillin (Sambrook et al., 1989). The clones containing recombinant plasmid with inserts in the right orientation were further selected by DNA sequencing using primers designed from positions on the vector upstream or downstream of the insert. 2.4. Overexpression and purification of T. cruzi AdoHcy hydrolase The transformed E. coli cells were grown in 2 YT medium containing 75 lg/ml ampicillin at 37 °C. Isopropyl-b-D -1-thiogalactopyranoside (IPTG) (1 mM) was added to induce the protein overexpression, and the temperature for cell growth was lowered to room temperature to avoid production of protein in an aggregated form. The cells were harvested after an additional 20 h of incubation. All of the subsequent protein purification procedures were carried out at 0–4 °C. The cell pellet was lysed in a Tris buffer (25 mM Tris, pH 7.2, containing 2 mM EDTA and 1 mg/ml lysozyme) followed by three freeze–thawing cycles. The cell-free lysate obtained from centrifugation was diluted with 1 volume of water and mixed with DEAE-cellulose at pH 7.2 (Sigma, St. Louis, MO). The protein was eluted from the DEAE-cellulose with a 100 mM potassium phosphate buffer (pH 7.2, containing 1 mM EDTA). The protein in the eluent was precipitated with 70% saturated (NH4 )2 SO4 . The collected pellet was dissolved buffer A (50 mM potassium phosphate buffer, pH 7.2, containing 1 mM EDTA) and applied to a size-exclusion gel filtration column (3 75 cm of Sephacryl S-300, Amersham–Pharmacia) pre-equilibrated with buffer A. Protein elution was monitored at 280 nm and fractions were analyzed for specific AdoHcy hydrolase activity (Henderson et al., 1992; Yang and Borchardt, 2000). The fractions containing AdoHcy hydrolase activity were pooled and further purified using a strong-anion exchange column (3 20 cm of Q-Sepharose, Amersham–Pharmacia) pre-equilibrated with buffer A. The fractions representing the first peak eluting from this weak-anion exchange column were collected, and (NH4 )2 SO4 precipitation followed by size-exclusion liquid chromatography as described above was used to obtain pure T. cruzi AdoHcy hydrolase. Protein concentrations were determined using Bradford reagents (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard (Sigma) (Bradford, 1976). The purity of the recombinant T. cruzi enzyme was assessed by SDS polyacrylamide gel electrophoresis. The T. cruzi apo AdoHcy hydrolase was prepared as previously described (Yuan et al., 1993).
2.5. Structural analysis of T. cruzi AdoHcy hydrolase The quaternary structure of the T. cruzi AdoHcy hydrolase was analyzed in 25 mM Tris buffer (pH 7.2) using a fast protein liquid chromatography (FPLC) system equipped with a size-exclusion column (Superdex 200 HR 10/30, Amersham–Pharmacia). The secondary structure of the enzyme was determined by circular dichroism (CD) spectroscopy. The CD spectra of 0.05 mg/ ml T. cruzi AdoHcy hydrolase in 25 mM Tris buffer (pH 7.2) were measured at 25 °C using a Jasco J-710 spectropolarimeter (Jasco, Tokyo, Japan) and a temperature-jacketed spectral cell with a pathlength of 1 cm. The apparent helical content was estimated using the program Contin, as previously described (Venyaminov and Yang, 1996; Yin et al., 2000a). 2.6. Assays of T. cruzi AdoHcy hydrolase activity Trypanosoma cruzi AdoHcy hydrolase activity was assayed in the synthetic direction by measuring the production rate of AdoHcy from Ado and Hcy using HPLC as previously described (Yuan et al., 1993). Quantitation of AdoHcy was accomplished using a UV detector set at 258 nm and a standard curve of AdoHcy (e258 ¼ 14; 900 M1 cm1 , Sigma) (Fasman, 1976). AdoHcy hydrolase activity in the hydrolytic direction was measured by monitoring the formation of the product Ado after its conversion to inosine using Ado deaminase. The conversion of Ado to inosine by Ado deaminase drives the AdoHcy hydrolase-catalyzed reaction in the hydrolytic direction (Spector, 1984; Turner et al., 2000). AdoHcy and inosine were separated on a C18 reversed-phase HPLC column (218TP54, Vydac, Hesperia, CA) equilibrated with 98% mobile phase A (10 mM 1-heptanesulfonic acid, 50 mM sodium phosphate, pH 3.2) and 2% mobile phase B (80% acetonitrile, 20% isopropanol). The elution gradient was 2–10% mobile phase B over 20 min at 1 ml/min flow rate. Inosine was monitored at 250 nm and quantified using a standard curve of inosine (e250 ¼ 12; 300 M1 cm1 , Sigma) (Fasman, 1976). 2.7. NADþ and NADH binding affinity The synthetic activity of T. cruzi AdoHcy hydrolase was measured using 1 lg/ml of the apo-enzyme, incubated with 1 mM Hcy and the indicated concentrations of NADþ in buffer A at 37 °C for 10 min. Ado (200 lM) was then added to start the reaction, and the samples were incubated at 37 °C for 10 min followed by addition of 50 lM HClO4 to quench the reaction. The data were fitted to a single-site binding model using the Origin Program (Microcal Software, Northampton, MA) as previously described to obtain the enzyme binding constant for NADþ , KdNAD (Yang and Borchardt, 2000).
N.B. Parker et al. / Experimental Parasitology 105 (2003) 149–158
Similarly, using 1 lg/ml apo-enzyme incubated with 100 lM NADþ and the indicated concentrations of NADH, the binding constant of KdNADH was calculated by fitting the data to the following equation: ½NADH KdNAD Activity ¼ Activitymax þ1 þ1 KdNADH ½NADþ
3. Results 3.1. Isolation and sequencing of the full T. cruzi AdoHcy hydrolase gene A survey of the active genes in T. cruzi performed by analyzing 5013 expressed sequence tags (ESTs) generated from a normalized epimastigote cDNA library has
153
shown homology between a segment of the T. cruzi cDNA and the L. donovani AdoHcy hydrolase gene (Porcel et al., 2000). The full-length L. donovani AdoHcy hydrolase cDNA was used as a template to generate 32 Plabeled PCR fragments by random priming. These fragments were used as probes for the isolation of the entire AdoHcy hydrolase gene locus from high density filters of an average 37 kb-insert genomic T. cruzi library (Hanke et al., 1996). On the basis of a genome size of the CL-Brener strain of around 45 Mb, the cosmid library would represent approximately 23 genome equivalents. Screening of the filters yielded 14 positive cosmid clones, three of which yielded sequencing data using a primer as described in Section 2. One of the positive clones, designated 62N9, was chosen for further analysis and was propagated and subjected to linear amplification sequencing. Our sequencing strategy produced the entire nucleotide sequence of the T. cruzi AdoHcy hydrolase
Fig. 2. AdoHcy hydrolase amino acid sequences alignment. Comparison of the deduced amino acid sequences of AdoHcy hydrolase from T. cruzi and other representative organisms was carried out using a program Lasergene from DNAStar (Madison, WI). GeneBank database Accession Nos. are M76556 (L. donovani); M61831 (human placental); NM017201 (rat). The dot ‘‘’’ indicates identical amino acid. ‘‘c’’ indicates residues involved in enzyme catalysis. ‘‘s’’ indicates residues involved in substrate binding. ‘‘n’’ indicates residues involved in cofactor NADþ binding. The solid line above and below the aligned sequences indicates significant non-conserved regions.
154
N.B. Parker et al. / Experimental Parasitology 105 (2003) 149–158
gene as well as several hundred nucleotides of 50 and 30 flanking sequences (Fig. 1). The entire nucleotide sequence was consolidated by sequencing of the complementary strand. The uninterrupted entire coding region of 1311 nt predicts a protein of 437 residues with a calculated molecular mass of 48,460 Da. The amino acid sequence corresponds to the proenzyme form of AdoHcy hydrolase, observed in all prokaryotic and eukaryotic species studied (Turner et al., 2000). Using the BLAST program at the website of the National Center for Biotechnology Information with default search parameters, the amino acid sequence of the T. cruzi AdoHcy hydrolase was compared to that of the enzyme from other organisms. The maximum gap alignment result shows that the T. cruzi enzyme is 70–73% identical to AdoHcy hydrolase from higher eukaryotes and 90–93% identical to AdoHcy hydrolase of other protozoa. For example, the overall sequence identity is 91 and 73% between the primary structure of the T. cruzi AdoHcy hydrolase and the corresponding enzymes from L. donovani and the human placenta, respectively (Fig. 2) (Coulter-Karis and Hershfield, 1989; Gomi et al., 1989; Henderson et al., 1992). The predicted amino acid sequence for the T. cruzi AdoHcy hydrolase is of the same length as that of L. donovani but five residues longer than that of mammalians. The sequence comparisons (Fig. 2) indicate that a greater similarity is seen between the T. cruzi and the L. donovani AdoHcy hydrolase than between the T. cruzi and the human or rat enzymes. The isoelectric points for the T. cruzi and the human AdoHcy hydrolase, as calculated on the basis of their amino acid sequences, differed significantly. However, pI values of 6.47 for the T. cruzi enzyme and 6.0 for the human enzyme show that both are acidic proteins. 3.2. Expression and purification of T. cruzi AdoHcy hydrolase The T. cruzi AdoHcy hydrolase gene was subcloned into an expression vector pPROK-1 at the EcoRI cloning site. The insertion was confirmed by restriction mapping with EcoRI. By automated DNA sequencing of the recombinant plasmid using primers from positions upstream and downstream of the insert, two clones with the insert in the right orientation were selected for protein overexpression. The T. cruzi AdoHcy hydrolase was expressed in the soluble form in the cell-free lysate as shown in Fig. 3 (lane 2). In the cell-free extract of E. coli, the recombinant enzyme represents approximately 30% of the total protein. After initial purification on a DEAE column, a crude product of about 70% purity was obtained (Fig. 3, lane 3). The purity was increased to 85% (Fig. 3, lane 4) after chromatography on a Sephacryl S-300 gel
Fig. 3. Coomassie blue-stained protein samples from each step of the purification. The recombinant T. cruzi AdoHcy hydrolase was purified as described in Section 2. The SDS acrylamide gel was loaded with protein molecular weight marker (lane 1); cell free extract of E. coli transfected with gene for T. cruzi AdoHcy hydrolase (lane 2); eluent from DEAE-cellulose (lane 3); fractions collected from size-exclusion chromatography (lane 4); fractions collected from ion-exchange chromatography (lane 5); and reconstituted and second size-exclusion column for purification of T. cruzi AdoHcy hydrolase (lane 6). The human recombinant AdoHcy hydrolase was used as a reference (lane 7).
filtration column. The next chromatography step using an anion-exchange column increased the purity to over 90%. The final step involving a second size-exclusion chromatography produced T. cruzi AdoHcy hydrolase of over 95% purity as shown by electrophoresis (Fig. 3, lane 6). About 20 mg of T. cruzi AdoHcy hydrolase can be routinely obtained from 1 liter of E. coli culture. In the presence of 100 lM NADþ at pH 7.2 and 37 °C, the recombinant T. cruzi enzyme catalyzes the interconversion of AdoHcy to adenosine (Ado) and homocysteine (Hcy) with a first-order rate constant of 1 s1 in the hydrolytic direction (AdoHcy ! Ado + Hcy) and a pseudo-first-order rate constant of 3 s1 in the synthetic direction (Ado + Hcy ! AdoHcy) in the presence of 1 mM Hcy. 3.3. Structure of T. cruzi AdoHcy hydrolase On SDS–PAGE, the recombinant T. cruzi enzyme migrated more slowly than the human AdoHcy hydrolase (Fig. 3, lane 7). The subunit molecular weight of the T. cruzi enzyme was estimated to be 48 3 kDa, which is similar to the predicted value (48.5 kDa) from the DNA sequence. Using size-exclusion semi-preparative FPLC, the T. cruzi enzyme elutes with an molecular weight of approximately 200 kDa, which is similar to that of human AdoHcy hydrolase (Fig. 4A). These results suggest that the T. cruzi enzyme exists as a tetramer similar to the human AdoHcy hydrolase. The secondary structure of the T. cruzi enzyme is nearly identical to that of the human AdoHcy hydrolase (Fig. 4B).
N.B. Parker et al. / Experimental Parasitology 105 (2003) 149–158
Fig. 4. Structural characterization of the purified T. cruzi AdoHcy hydrolase. Upper panel, chromatography of T. cruzi (solid line) and human AdoHcy hydrolase (dash-dot line) analyzed using a size-exclusion FPLC column as described in Section 2. Lower panel, CD spectra comparison of T. cruzi (solid circle) and human AdoHcy hydrolase (open circle).
3.4. Substrate kinetic properties of the recombinant T. cruzi AdoHcy hydrolase Fig. 5 shows that the activity of T. cruzi AdoHcy hydrolase is dependent on the concentration of the cofactor NADþ . When these data were fitted to a one-site binding model, a Kd of 1.3 0.1 lM was estimated for NADþ . Binding of NADH to T. cruzi AdoHcy hydrolase produced an inactive form of the enzyme. The Kd for NADH was estimated to be 104 7 nM from the data shown in Fig. 5 as described in Section 2. These data indicate that the affinity of T. cruzi AdoHcy hydrolase for NADH is about 13 times higher than that for NADþ .
4. Discussion Active AdoMet-dependent methyltransferases (e.g., guanine N 7 -methyltransferase, EC 2.1.1.33, and d24ð25Þ sterol methyltransferase, EC 2.1.1.41) have been identified in parasites, including Encephalitozoon cuniculi, L. donovani, Pneumocystis carinii, Trichostrongylus colubriformis, and T. cruzi (Frandsen and Bone, 1988; Hasne and Lawrence, 1999; Hausmann et al., 2002; Urbina et al., 1997; Urbina et al., 1995). Methylations of the
155
Fig. 5. The relationship between the enzyme activity and the concentration of NADþ and NADH in the reaction solution. The activities of T. cruzi AdoHcy hydrolase reconstituted in the apo-form were assayed in the synthetic direction at different concentrations of NADþ and NADH. Curves are results from the model fitting described in Section 2.
type catalyzed by guanine N 7 -methyltransferase and d24ð25Þ -sterol methyltransferase have been inferred to occur in T. cruzi from the presence of 50 -end methylated SL RNA and methylated sterols (Ullu and Tschudi, 1995; Urbina et al., 1996). Metabolism of AdoHcy, the product inhibitor of AdoMet-dependent methyltransferases, has been shown to be catalyzed by AdoHcy hydrolase produced in the parasites L. donovani, P. falciparum, and T. vaginalis (Creedon et al., 1994; Henderson et al., 1992; Minotto et al., 1998). As yet, AdoHcy hydrolase activity in T. cruzi has not been reported. However, sequencing data generated from a T. cruzi epimastigote normalized cDNA library, as part of the T. cruzi genome project, have presented seven clones with a cDNA sequence similar to part of the L. donovani AdoHcy hydrolase gene (Porcel et al., 2000). Using a computer alignment program (BLAST 2.0, Washington University, St. Louis, MO) to compare the T. cruzi AdoHcy hydrolase gene from our study to the seven clones in the cDNA library, we found that all the cDNA sequences (ranging from 237 to 549 bp) were covered by the full-length AdoHcy hydrolase gene from the genomic library of T. cruzi DNA with 96–100% identities to
156
N.B. Parker et al. / Experimental Parasitology 105 (2003) 149–158
corresponding regions (Korf and Gish, 2000). This comparison indicates that the T. cruzi AdoHcy hydrolase gene is expressed in this parasite. The amino acid sequences of AdoHcy hydrolase from mammalians and parasites have a high level of conservation during evolution. For example, the T. cruzi AdoHcy hydrolase sequence exhibits a high positive similarity score with the L. donovani sequence (93%) and with human and rat sequences (81%) by the Gapped BLAST program (Altschul et al., 1997). X-ray crystal structures of the human and rat AdoHcy hydrolase suggest certain amino acid residues to be involved in substrate and cofactor binding and in catalysis (Turner et al., 2000). The critical roles of these residues have also been confirmed by site-directed mutagenesis (unpublished data) and computational modeling (Hu et al., 2001). As shown in Fig. 2, most of these residues are conserved among the AdoHcy hydrolase sequences from T. cruzi, L. donovani, human, and rat. The one exception is residue 56, which is a Gln in the T. cruzi and L. donovani enzymes and Glu in the human and rat enzymes. With these highly conserved sequences, it is not surprising that the T. cruzi AdoHcy hydrolase has quaternary and secondary structures similar to those of the human enzyme (Fig. 4). However, the variable regions in the sequences apparently produce some differences in the properties of the T. cruzi and human AdoHcy hydrolase. One difference is the charge characteristics of the proteins. For example, human AdoHcy hydrolase has a calculated pI of 6.3, and it binds to a weak anion exchange column in 50 mM phosphate buffer at pH 7.2. In contrast, under the same conditions T. cruzi AdoHcy hydrolase, with a predicted pI of 6.5, does not bind to this weak anion exchange column. It is possible that the difference in their experimental pI values in solution may be significantly larger than their calculated pI values. If potent inhibitors of a parasite AdoHcy hydrolase (e.g., T. cruzi) with minimal effects on the human enzyme could be designed, these types of compounds might have clinical potential as antiparasitic agents. A difference in the properties of the parasite and human AdoHcy hydrolase that might be exploited for the drug design purpose is the binding of the cofactor NADþ . It is well known that mammalian AdoHcy hydrolase binds tightly one molecule of the cofactor per enzyme subunit (Palmer and Abeles, 1979). The enzyme is positively cooperative in binding NADþ (Gomi et al., 1989). The Kd value of human AdoHcy hydrolase is 120 20 nM for both NADþ and NADH (Yang and Borchardt, 2000). Release of the cofactors from the human enzyme is not normally observed due to the very slow dissociation rates (Gomi et al., 1989). In contrast, the T. cruzi enzyme binds NADþ (Kd ¼ 1:3 lM) more loosely to the four sites of the tetrameric enzyme. Similar results have been reported for the L. donovani enzyme (Yang and
Borchardt, 2000). However, the T. cruzi AdoHcy hydrolase has an affinity (Kd ¼ 104 nM) for NADH that is similar to that of the human enzyme but 20 times lower than the Kd for the L. donovani enzyme (Yang and Borchardt, 2000). From the sequence comparison, we observed three non-conserved regions close to the cofactor binding sites (Fig. 2). These regions may modulate the affinity of AdoHcy hydrolase for the cofactor. Further side-directed mutagenesis in these regions is needed to test our hypothesis. The differences found in the binding properties of T. cruzi AdoHcy hydrolase relative to those of the human enzyme for NADþ are being exploited in our laboratory for the design of antitrypanosoma drugs that selectively inhibit T. cruzi AdoHcy hydrolase.
Acknowledgments This work was supported in part by a grant from the National Institutes of Health (GM29332) and a Postdoctoral Fellowship to D.H.Y. from the American Heart Association Heartland Affiliate (9920522Z). We thank Dr. Jinsong Zhang, Dr. Yongbo Hu, and Ms. Mengmeng Wang, The University of Kansas, and Dr. P. Lynne Howell, Hospital for Sick Children, Toronto, Canada, for insightful discussions. We thank Clark Bloomer, Biotechnology Support Facility at University of Kansas Medical Center, for his work on DNA sequencing, and Nancy Harmony for proofreading and editing the manuscript.
References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25, 3389–3402. Araujo-Jorge, T.C., 1999. Biology and ultra-structure of Trypanosoma cruzi: a 90-year old challenge for scientists. Memorias do Instituto Oswaldo Cruz 94, 131–134. Avila, J.L., Avila, A., Polegre, M.A., Marquez, V.E., 1997. Specific inhibitory effect of 3-deazaneplanocin A against several Leishmania mexicana and L. braziliensis strains. American Journal of Tropical Medicine and Hygiene 57, 407–412. Bacchi, C.J., Goldberg, B., Garofalo-Hannan, J., Rattendi, D., Lyte, P., Yarlett, N., 1995. Fate of soluble methionine in African trypanosomes: effects of metabolic inhibitors. The Biochemical Journal 309, 737–743. Bethin, K.E., Cimato, T.R., Ettinger, M.J., 1995. Copper binding to mouse liver S-adenosylhomocysteine hydrolase and the effects of copper on its levels. Journal of Biological Chemistry 270, 20703– 20711. Bitonti, A.J., Baumann, R.J., Jarvi, E.T., McCarthy, J.R., McCann, P.P., 1990. Antimalarial activity of a 4Õ,5Õ-unsaturated 5Õ-fluoroadenosine mechanism-based inhibitor of S-adenosyl-L -homocysteine hydrolase. Biochemical Pharmacology 40, 601–606. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the
N.B. Parker et al. / Experimental Parasitology 105 (2003) 149–158 principle of protein–dye binding. Analytical Biochemistry 72, 248–254. Caggese, C., Ragone, G., Barsanti, P., Moschetti, R., Messina, A., Massari, S., Caizzi, R., 1997. The S-adenosyl-L -homocysteine hydrolase of Drosophila melanogaster: identification, deduced amino acid sequence and cytological localization of the structural gene. Molecular and General Genetics 253, 492–498. Chiang, P.K., 1998. Biological effects of inhibitors of S-adenosylhomocysteine hydrolase. Pharmacology and Therapeutics 77, 115– 134. Coulter-Karis, D.E., Hershfield, M.S., 1989. Sequence of full length cDNA for human S-adenosylhomocysteine hydrolase. Annals of Human Genetics 53, 169–175. Creedon, K.A., Rathod, P.K., Wellems, T.E., 1994. Plasmodium falciparum S -adenosylhomocysteine hydrolase. cDNA identification, predicted protein sequence, and expression in Escherichia coli. Journal of Biological Chemistry 269, 16364–16370. Fasman, G.D., 1976. In: Fasman, G.D. (Ed.), Handbook of Biochemistry and Molecular Biology: Nucleic Acid II. CRC Press, Cleveland, OH, pp. 97–120. Frandsen, J.C., Bone, L.W., 1988. Derivatives of epinephrine, norepinephrine, octopamine and histamine formed by homogenates of Trichostrongylus colubriformis, a nematode parasite of ruminants. Comparative Biochemistry and Physiology C 91, 385–387. Freistadt, M.S., Cross, G.A., Robertson, H.D., 1988. Discontinuously synthesized mRNA from Trypanosoma brucei contains the highly methylated 5Õ cap structure, m7GpppA*A*C(2Õ-O)mU*A. Journal of Biological Chemistry 263, 15071–15075. Gomi, T., Takata, Y., Fujioka, M., 1989. Rat liver S-adenosylhomocysteinase. Spectrophotometric study of coenzyme binding. Biochimica et Biophysica Acta 994, 172–179. Hanke, J., Sanchez, D.O., Henriksson, J., Aslund, L., Pettersson, U., Frasch, A.C., Hoheisel, J.D., 1996. Mapping the Trypanosoma cruzi genome: analyses of representative cosmid libraries. Biotechniques 21, 686–688, 690-693. Hasne, M.P., Lawrence, F., 1999. Characterization of prenylated protein methyltransferase in Leishmania. The Biochemical Journal 342 (Part 3), 513–518. Hausmann, S., Vivares, C.P., Shuman, S., 2002. Characterization of the mRNA capping apparatus of the microsporidian parasite Encephalitozoon cuniculi. Journal of Biological Chemistry 277, 96– 103. Henderson, D.M., Hanson, S., Allen, T., Wilson, K., Coulter-Karis, D.E., Greenberg, M.L., Hershfield, M.S., Ullman, B., 1992. Cloning of the gene encoding Leishmania donovani S -adenosylhomocysteine hydrolase, a potential target for antiparasitic chemotherapy. Molecular and Biochemical Parasitology 53, 169–183. Hu, Y., Yang, X., Yin, D.H., Mahadevan, J., Kuczera, K., Schowen, R.L., Borchardt, R.T., 2001. Computational characterization of substrate binding and catalysis in S-adenosylhomocysteine hydrolase. Biochemistry 40, 15143–15152. Kirchhoff, L.V., 1993. American trypanosomiasis (ChagasÕ disease)—a tropical disease now in the United States. The New England Journal of Medicine 329, 639–644. Kitade, Y., Kozaki, A., Gotoh, T., Miwa, T., Nakanishi, M., Yatome, C., 1999. Synthesis of S-adenosyl-L -homocysteine hydrolase inhibitors and their biological activities. Nucleic Acids Symposium Series 42, 25–26. Korf, I., Gish, W., 2000. MPBLAST: improved BLAST performance with multiplexed queries. Bioinformatics 16, 1052–1053. Merta, A., Aksamit, R.R., Kasir, J., Cantoni, G.L., 1995. The gene and pseudogenes of rat S-adenosyl-L -homocysteine hydrolase. European Journal of Biochemistry 229, 575–582. Minotto, L., Ko, G.A., Edwards, M.R., Bagnara, A.S., 1998. Trichomonas vaginalis: expression and characterisation of recombinant S-adenosylhomocysteinase. Experimental Parasitology 90, 175–180.
157
Nakajima-Shimada, J., Hirota, Y., Aoki, T., 1996. Inhibition of Trypanosoma cruzi growth in mammalian cells by purine and pyrimidine analogs. Antimicrobial Agents and Chemotherapy 40, 2455–2458. Palmer, J.L., Abeles, R.H., 1979. The mechanism of action of Sadenosylhomocysteinase. Journal of Biological Chemistry 254, 1217–1226. Persson, K., Aslund, L., Grahn, B., Hanke, J., Heby, O., 1998. Trypanosoma cruzi has not lost its S-adenosylmethionine decarboxylase: characterization of the gene and the encoded enzyme. Biochemical Journal 333, 527–537. Phelouzat, M.A., Lawrence, F., Moulay, L., Borot, C., Schaeverbeke, J., Schaeverbeke, M., Robert-Gero, M., 1992. Leishmania donovani: antagonistic effect of S-adenosyl methionine on ultrastructural changes and growth inhibition induced by sinefungin. Experimental Parasitology 74, 177–187. Porcel, B.M., Tran, A.N., Tammi, M., Nyarady, Z., Rydaker, M., Urmenyi, T.P., Rondinelli, E., Pettersson, U., Andersson, B., Aslund, L., 2000. Gene survey of the pathogenic protozoan Trypanosoma cruzi. Genome Research 10, 1103–1107. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Spector, T., 1984. Progress curve analysis of adenosine deaminasecatalyzed reactions. Analytical Biochemistry 138, 242–245. Turner, M.A., Yang, X., Yin, D., Kuczera, K., Borchardt, R.T., Howell, P.L., 2000. Structure and function of S-adenosylhomocysteine hydrolase. Cell Biochemistry and Biophysics 33, 101–125. Ullu, E., Tschudi, C., 1995. Accurate modification of the trypanosome spliced leader cap structure in a homologous cell-free system. Journal of Biological Chemistry 270, 20365–20369. Urbina, J.A., 1999. Parasitological cure of Chagas disease: is it possible? Is it relevant? Memorias do Instituto Oswaldo Cruz 94, 349–355. Urbina, J.A., Visbal, G., Contreras, L.M., McLaughlin, G., Docampo, R., 1997. Inhibitors of delta24(25) sterol methyltransferase block sterol synthesis and cell proliferation in Pneumocystis carinii. Antimicrobial Agents and Chemotherapy 41, 1428–1432. Urbina, J.A., Vivas, J., Lazardi, K., Molina, J., Payares, G., Piras, M.M., Piras, R., 1996. Antiproliferative effects of delta 24(25) sterol methyl transferase inhibitors on Trypanosoma (Schizotrypanum) cruzi: in vitro and in vivo studies. Chemotherapy 42, 294– 307. Urbina, J.A., Vivas, J., Visbal, G., Contreras, L.M., 1995. Modification of the sterol composition of Trypanosoma (Schizotrypanum) cruzi epimastigotes by delta 24(25)-sterol methyl transferase inhibitors and their combinations with ketoconazole. Molecular and Biochemical Parasitology 73, 199–210. Venyaminov, S.Y., Yang, J.T., 1996. Circular Dichroism and the Conformational Analysis of Biomolecules. Plenum Press, New York, NY. pp. 69–107. Whaun, J.M., Miura, G.A., Brown, N.D., Gordon, R.K., Chiang, P.K., 1986. Antimalarial activity of neplanocin A with perturbations in the metabolism of purines, polyamines and S-adenosylmethionine. The Journal of Pharmacology and Experimental Therapeutics 236, 277–283. WHO, 1991. Control of Chagas disease. Report of a WHO Expert Committee. World Health Organization Technical Report Series 811, pp. 1–95. Yang, X., Borchardt, R.T., 2000. Overexpression, purification, and characterization of S-adenosylhomocysteine hydrolase from Leishmania donovani. Archives of Biochemistry and Biophysics 383, 272– 280. Yin, D., Sun, H., Ferrington, D.A., Squier, T.C., 2000a. Closer proximity between opposing domains of vertebrate calmodulin following deletion of Met(145)–Lys(148). Biochemistry 39, 10255– 10268.
158
N.B. Parker et al. / Experimental Parasitology 105 (2003) 149–158
Yin, D., Yang, X., Borchardt, R.T., Yuan, C.-S., 2000b. Mechanismbased S-adenosyl-L -homocysteine hydrolase inhibitors in the search for broad-spectrum antiviral agents. In: Torrence, P.F. (Ed.), Biomedical Chemistry Applying Chemical Principles to the Understanding and Treatment of Disease. Wiley, New York, NY, pp. 41–71.
Yuan, C.S., Yeh, J., Squier, T.C., Rawitch, A., Borchardt, R.T., 1993. Ligand-dependent changes in intrinsic fluorescence of Sadenosylhomocysteine hydrolase: implications for the mechanism of inhibitor-induced inhibition. Biochemistry 32, 10414– 10422.