Evolution of Cryptosporidium parvum Lactate Dehydrogenase from Malate Dehydrogenase by a Very Recent Event of Gene Duplication
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分子生物学进展 2004年第3期
* Laboratoire de Biophysique Moléculaire, Institut de Biologie Structurale CEA-CNRS-UJF, Grenoble, France
Department of Veterinary Pathobiology, Texas A&M University, College Station
Department of Veterinary Pathobiology, University of Minnesota, St. Paul
E-mail: g3hu@cvm.tamu.edu.
Abstract
We have expressed the L-lactate dehydrogenase (LDH) and L-malate dehydrogenase (malDH) genes from the apicomplexan Cryptosporidium parvum (CpLDH1 and CpMalDH1) as maltose-binding protein (MBP) fusion proteins in Escherichia coli. The substrate specificities, enzymatic kinetics, and oligomeric states of these two parasite enzymes have been characterized. By taking advantage of recently completed and ongoing apicomplexan genome sequencing projects, we identified additional MalDH genes from Plasmodium spp., Toxoplasma gondii, and Eimeria tenella that were previously unavailable. All apicomplexan MalDHs appeared to be cytosolic and no organellar homologs were identified from the completely sequenced P. falciparum genome and other ongoing apicomplexan genome-sequencing projects. Using these expanded apicomplexan LDH and MalDH sequence databases, we reexamined their phylogenetic relationships and reconfirmed their relationship to -proteobacterial MalDHs. All LDH and MalDH enzymes from apicomplexans were monophyletic within the LDH-like MalDH group (i.e., MalDH resembling LDH) as a sister to -proteobacterial MalDHs. All apicomplexan LDHs, with the exception of CpLDH1, formed a separate clade from their MalDH counterparts, indicating that these LDHs were evolved from an ancestral apicomplexan MalDH by a gene duplication coupled with functional conversion before the expansion of apicomplexans. Finally, CpLDH1 was consistently placed together with CpMalDH1 within the apicomplexan MalDH cluster, confirming an early working hypothesis that CpLDH1 was probably evolved from the same ancestor of CpMalDH1 by a very recent gene duplication that occurred after C. parvum diverged from other apicomplexans.
Key Words: Apicomplexa ? Cryptosporidium parvum ? malate dehydrogenase ? lactate dehydrogenase ? phylogeny ? evolution ? gene duplication
Introduction
L-malate dehydrogenase (L-MalDH) and L-lactate dehydrogenase (L-LDH) belong to the superfamily of 2-ketoacid:NAD(P)–dependent dehydrogenases that catalyze the reversible conversion of 2-hydroxyacids to the corresponding 2-ketoacids. MalDHs and LDHs (all MalDHs and LDHs herein refer to L-MalDH and L-LDHs, respectively) have been purified and characterized from a wide variety of organisms. MalDHs were found to be widespread over the three domains of life, whereas LDHs are solely present in Eukarya and Bacteria. Recent phylogenetic reconstructions have shown that two ancestral paralogous gene duplications explain the main functional distribution and evolution of MalDH and LDH (Madern 2002). These duplications have lead to the existence of three major clades of enzymes that differ strongly in their structural and biochemical features (Madern 2002). The first clade includes the two well-described subgroups of mitochondrial and cytosolic dimeric MalDHs (Roger, Morrison, and Sogin 1999). The others are the LDH-like MalDHs (i.e., MalDH resembling LDH) and LDHs. LDH-like MalDHs and LDHs have a similar tetrameric architecture based on crystallographic analysis (Richard et al. 2000; Lee et al. 2001; Dalhus et al. 2002). Therefore, in contrast to previous scenarios of molecular evolution between MalDHs and LDHs (described without any knowledge of the LDH-like MalDHs), Madern (2002) proposed that LDHs were not the result of a primary ancestral duplication from MalDHs (in the dimeric state), but were very likely selected from an ancient gene duplication with LDH-like MalDHs.
According to the general numbering scheme for LDH and MalDH enzymes (Eventoff et al. 1977), residues at position 102 are the most important in determining substrate specificity (Wilks et al. 1988; Nicholls et al. 1992; Cendrin et al. 1993), which is typically a charged arginine in MalDHs and an uncharged residue (mostly glutamine) in LDHs, respectively. Mutagenesis experiments have clearly demonstrated that single amino acid change from glutamine to arginine at the position 102 might change LDH to MalDH (Wilks et al. 1988; Goward and Nicholls 1994; Golding and Dean 1998), although functional conversion from MalDH to LDH has been much less successful by replacing arginine with an uncharged residue (Cendrin et al. 1993; Boernke et al. 1995; Wu et al. 1999). However, besides the two ancestral gene duplications mentioned above, recent functional changes between MalDH and LDH appeared to rarely occur in the nature. There has been no evidence that the LDH lineage contains any MalDH enzymes. On the other hand, only a few types of LDHs were intermixed within the various MalDH groups. For example, trichomonad LDHs were found linked with the cytosolic dimeric MalDH (Cazzulo Franke et al. 1999), whereas apicomplexan LDHs were rooted with LDH-like MalDH (Wu et al. 1999; Zhu and Keithly 2002). The trichomonad LDHs probably evolved from their cytosolic dimeric MalDH in recent events of gene duplication. Apicomplexan LDH and MalDH enzymes were found to be monophyletic to the -proteobacterial LDH-like MalDHs, suggesting that these parasite enzymes might be acquired from an -proteobacterium by a lateral gene transfer (LGT) event or by retaining the bacterial LDH-like MalDHs during the primary endosymbiosis that gave rise to mitochondria. In agreement with these individual observations, the general molecular evolution pathway of MalDHs and LDHs seems to be polarized from high to low stringency of substrate recognition, leading to functional changes of MalDHs towards LDHs without any evidence for the reverse phenomenon (Madern 2002).
Among apicomplexans, early studies have shown that Toxoplasma gondii possess two LDH genes that are differentially expressed during their complex life cycle (Yang and Parmley 1997) Only one LDH gene from Plasmodium falciparum was cloned and intensively studied (Simmons et al. 1985), although, recently, genome projects revealed the presence of another putative, but a highly divergent, LDH gene in this parasite (i.e., GenBank accession number CAD52400). Analysis of the completely sequenced P. falciparum genome and the ongoing T. gondii genome project supports the belief that both organisms only have these two LDH genes. Furthermore, a single MalDH gene encoding a cytosolic enzyme was recently identified from each of these two apicomplexan genomes. In contrast, only one each of the putative MalDH and LDH genes from Cryptosporidium parvum has been previously reported, and no additional MalDH/LDH genes could be found within its completed genome sequence. In this study, we expressed the both C. parvum MalDH and LDH in Escherichia coli as maltose-binding protein (MBP) fusion proteins to correctly assess their respective enzymatic function and oligomeric states in solutions. With the expanded MalDH and LDH sequences from both apicomplexans and -proteobacteria, new phylogenetic reconstructions have reconfirmed our early observations that these enzymes are linked to the LDH-like MalDHs (Madern 2002; Zhu and Keithly 2002). They also revealed that all apicomplexan LDH-like MalDHs and LDH enzymes were monophyletic, which suggested that most apicomplexan LDH-like MalDHs and LDHs evolved from a common ancestor by a gene duplication before the evolutionary expansion of apicomplexans. More interestingly, C. parvum LDH gene appeared to evolve from its cytosolic partner, LDH-like MalDH (independently from those of other apicomplexan LDHs), by a very recent gene duplication that occurred after this organism diverged from other apicomplexans.
Materials and Methods
Expression and Biochemical Characterization of CpMalDH1 and CpLDH1 Enzymes
Two genes encoding putative MalDH and LDH enzymes were previously identified from C. parvum, which were designated to CpMalDH1 and CpLDH1, respectively (Zhu and Keithly 2002). Their nucleotide sequences were deposited in GenBank under the accession numbers AF274310 (CpLDH1) and AY334274 (CpMalDH1). Their initial annotations were based on the key amino acid at the active site loop in which CpMalDH1 possessed an arginine, and CpLDH1 had an uncharged glycine that differed from the glutamine found in the majority of LDHs. Data mining the nearly completely sequenced C. parvum genome indicated that CpMalDH1 and CpLDH1 were the only 2-ketoacid dehydrogenase homologs possessed by this parasite.
The entire open reading frames (ORFs) of the CpMalDH1 and CpLDH1 genes were amplified by PCR using high-fidelity AccuTaq DNA polymerase (Sigma Chemical Co., St Louis, Mo.) for cloning into a pMAL-c2x vector (New England Biolabs, Beverly, Mass.) at the EcoRI restriction site. Because the cpMalDH gene contained internal EcoRI sites, a strategy to avoid post-PCR digestion of amplicons with EcoRI was employed (fig. 1): first, oligonucleotide linkers "aattc" (long type) or "c" (short type) was added to each of the sense and antisense primers; second, the long type sense primers were paired only with the short antisense ones (and vice versa) in PCR to produce two types of amplicons for each of the two genes; third, all PCR products were phosphorylated with T7 DNA kinase, and the kinase was then removed using a DNA digestion cleanup kit (Qiagen, Valencia, Calif); and fourth, the same amounts of the separately amplified PCR products for each gene were mixed together and denatured by heating at 100°C for 10 min and slowly cooling down to room temperature within a heat block. Theoretically, 25% of the reannealed amplicons would contain the phosphorylated 5'-aatt overhanging at both ends, which could be subsequently ligated into pMAL-c2x vector (fig. 1).
FIG. 1. Amplification of CpMalDH1 and CpLDH1 open reading frames (ORFs). Because CpMalDH1 ORF contained an internal EcoRI restriction site, a strategy to avoid post-PCR digestion of EcoRI by directly creating 5'-aatt overhanging ends was employed during the amplification and engineering of CpMDH1 and CpLDH1 genes into the pMAL-c2x at the EcoRI restriction site. Letters in lowercase represent artificially added nucleotide linkers
After ligation and transformation into E. coli competent cells, bacterial clones were subjected to direct PCR screening to determine the orientation of their inserts. Plasmids containing correctly oriented CpMalDH1 or CpLDH1 inserts were sequenced to confirm their identities and transformed into an E. coli Rosetta strain (Novagen, Madison, Wis.) for the expression of MBP-fusion proteins. Bacterial transformants were allowed to grow at 37°C until OD590 reached 0.4 to 0.6, which was followed by the addition of 0.5 mM IPTG and subsequent growth at 20°C for 6 h. The bacteria were collected by centrifugation and disrupted by sonication at 4°C in a binding buffer containing PMSF, EDTA, and pepstatin. Recombinant CpMalDH1 and CpLDH1 proteins were purified from supernatants using an amylose resin-based affinity chromatography according to the manufacturer's instructions. In some cases, the fusion proteins were further purified using ion exchange chromatography (Mono-Q resin). A portion of the purified fusion proteins was subjected to factor Xa digestion to separate the MBP-tag and parasite proteins. The purity of fusion proteins was analyzed in 10% SDS-PAGE gels, and their biochemical properties were characterized as described below.
The enzymatic activity of recombinant MBP-CpMalDH1 or MBP-CpLDH1 was measured in 50 mM Tris HCl buffer (pH 8.0) containing 0.2 mM NADH and
0.3 mM oxaloacetate or pyruvate, respectively. NADH oxidation by both enzymes was monitored at 340 nm on a Beckman DU7400 spectrophotometer at 25°C. The Km and Vmax values of the fusion proteins were determined for NADH, pyruvate, and oxaloacetate, respectively. Using the same assays, we also tested whether CpLDH1 activity could be inhibited by chloroquine, a potent inhibitor of P. falciparum LDH (Read et al. 1999).
The oligomeric states of these two parasite fusion proteins were analyzed by a size exclusion chromatography (Sephacryl S200). Sedimentation velocity analysis was performed on the recombinant proteins. This method has been shown to be very efficient for determining unambiguously the oligomeric state of LDH-like MalDH (Madern et al. 2000). Experiments were performed on a Beckman XLA analytical ultracentrifuge, equipped with a UV scanning system, using a four hole AN-60 Ti rotor with double centerpieces of 1.20 cm path-length. In a typical experiment, 200 absorbance profiles for each sample were recorded at 42,000 rpm at 20°C. The wavelengths were chosen according to the characteristics of the samples. The scan profiles were analyzed with the SEDFIT program (Schuck 2000) using the continuous sedimentation (or mass) distribution option. The partial specific volume of MBP-CpLDH1 (2 = 0.7419) and MBP-CpMalDH1 (2 = 0.7439) were determined from their primary sequence. The coefficients, S20,W, were corrected for temperature and viscosity using tabulated viscosity and density . Theoretical S values were calculated using the predicRHDC software.
Phylogenetic Reconstructions of Apicomplexan MalDH and LDH Enzymes
The cloning and sequencing of CpMalDH1 and CpLDH1 enzymes, as well as those of P. falciparum and T. gondii LDHs were previously described (Zhu and Keithly 2002). However, only one MalDH (i.e., CpMalDH1) sequence was previously available (Zhu and Keithly 2002). Therefore, the phylogenetic relationships of apicomplexan MalDHs and LDHs were not fully studied before. More recently, with the completion of P. falciparum (Haematozoa) genome (http://www.plasmodb.org) and the rapid progress of the ongoing T. gondii (cyst-forming Coccidia) (http://www.tigr.org/tdb/e2k1/tga1) and Eimeria tenella (intestinal Coccidia) (http://www.sanger.ac.uk/Projects/E_tenella) genome-sequencing projects, we were able to retrieve several apicomplexan MalDH genes from their corresponding databases. Because apicomplexan MalDH/LDH enzymes have shown to be evolutionarily related to the -proteobacterial LDH-like MalDHs (Zhu and Keithly 2002), a number of MalDH genes from this group of bacteria were also retrieved from the GenBank and used as an outgroup in the phylogenetic analysis. In total, seven apicomplexan LDH, five apicomplexan MalDH, and 13 -proteobacterial MalDH enzymes were included in the phylogenetic reconstructions.
All MalDH/LDH protein sequences were conceptually translated from their corresponding genes and aligned together using the MacVector version 7.1.1 program (Acceryl, Inc.). Aligned sequences were visually inspected to correct apparent mistakes. Finally, 278 unambiguously aligned amino acid positions were used in the analysis. The Tree-Puzzle version 5.0 program (Strimmer and von Haeseler 1996) was first used to generate approximate quartet likelihood trees with 10,000 puzzling steps. Parameters were calculated from the topology of a neighbor-joining tree and the amino acid frequencies (f) were estimated from data sets using a JTT model of amino acid substitution with the consideration of rate heterogeneity; that is, the fraction of invariance and four-rate gamma distributions (JTT–f + + Inv.). Parameters established from puzzling analysis were then applied to a true maximum-likelihood (ML) analysis using a ProML program in the PHYLIP version 3.6a2 package (Felsenstein 1993) with sequence input order randomized and global rearrangements enabled during the tree search. Similarly, ML bootstrapping analysis was performed using the ProML program from 100 replicates generated by the SeqBoot program. In addition, phylogenetic trees were also reconstructed with the JTT model of amino acid substitutions and the same rates of heterogeneity (JTT–f + + Inv.) using a Bayesian inference (BI) method (Huelsenbeck and Ronquist 2001). A total of 500,000 generations of searches were performed with four chains simultaneously running. Stable ML values were quickly reached before 10,000 generations of searches, indicating that the Markov chain Monte Carlo (MCMC) application was allowed to run for sufficient generations. Posterior probabilities at tree nodes were obtained by calculating the consensus tree from the best 4,500 BI trees using a 50% majority ruling method.
Results and Discussions
Characterization of CpMalDH1 and CpLDH1 Oligomeric States
Our phylogenetic analysis (see below) and previous reconstructions have shown that apicomplexan MalDHs and LDHs are rooted within the group of LDH-like MalDHs (Madern 2002; Zhu and Keithly 2002). Crystallographic structures of three LDH-like MalDH have revealed their tetrameric organization (dimer of dimers) (Richard et al. 2000; Lee et al. 2001; Dalhus et al. 2002). According to this, our aim was to determine more precisely the respective oligomeric state of CpMalDH1 or CpLDH1 (both expected to be tetrameric). Unfortunately, we encountered some technical difficulties to efficiently separate the CpMalDH1 or CpLDH1 portion from the MBP-tag by digesting fusion proteins with factor Xa. We decided therefore to record data using pure MBP-fusion proteins.
In a first attempt, we analyzed the oligomeric state of both MBP-fusions by size exclusion chromatography using a well-calibrated column. In contrast to our expectations, recombinant active MBP-fusion proteins were recorded with an elution volume higher than that estimated for tetrameric MBP-fusions, indicating they were not tetrameric. To get more accurate data, we have recorded the enzyme properties using analytical centrifugation (AUC).
Pure tetrameric MBP-CpMalDH1 and MBP-CpLDH1 would migrate in an AUC experiment as single species with theoritical sedimentation coefficient of S20,W = 12.5 S corresponding to spherical proteins of 316 kDa. In contrast to our expectation, a sedimentation velocity analysis of MBP-CpMalDH1 protein identified two main species with lower apparent sedimentation coefficiencies. The data (not shown) were well fitted with a similar amount of slightly elongated dimeric and monomeric state of MBP-CpMalDH1 with S20,W of 6.2 S and 3.9 S, respectively. A tiny fraction of bigger species (< 2%) was also observed that could have been caused by a nonspecific aggregation rather than by a very small amount of tetrameric molecules. The two major species displayed apparent molecular weights at 73 and 156 kDa, respectively, which were close to the theoretical values of a monomer (76 kDa) and a dimer (153 kDa) for the MBP-CpMalDH1 fusion. Analysis of the structural information available for LDH-like MalDHs shows that a distortion of the N-terminus might propagate toward the region that controls the association between monomers. In our case, the data suggest that MBP-tag fused at the N-terminal part of CpMalDH has very likely induced some structural reorganizations (because of steric hindrance) that has drastically modified the folding and association pathway, allowing it to trap intermediates that exists during this process. Using a recombinant enzyme from Haloarcula marismortui, dimeric intermediate species that assemble to make a tetrameric LDH-like MalDH have been demonstrated to be active (Madern et al. 2000). According to this observation, the purified MBP-CpMalDH1 that contains dimeric species would be satisfactory to determine enzymatic properties.
Similarly, two species of MBP-CpLDH1 protein (instead of a single species of 317 kDa with S20,W = 12.5 S) were also present in solution with apparent S20,W of 6.0 S and 4.2 S, respectively. Their molecular weights, determined by the mass distribution option of Sedfit, were 77 and 134 kDa, respectively, which agreed with calculated molecular weights of MBP-CpLDH1 (i.e., monomer Mr = 77 kDa and a dimer Mr = 153 kDa). As discussed previously, the same analysis and conclusion holds for MBP-CpLDH1. All of these observations suggested a monomer-dimer equilibrium for both parasite fusion proteins in solution.
Enzymatic Properties of CpMalDH1 and CpLDH1
It has been shown that P. falciparum and T. gondii LDHs differ from other organisms (including their hosts) by possessing a 5-aa insertion at their catalytic sites, which could imply a unique mode of action of these enzymes. The 5-aa insertion is also present in recently cloned E. tenella, E. acervulina, and E. maxima LDHs (fig. 2). However, this unique insertion is not present in CpLDH1 or any of the apicomplexan MalDHs. Therefore, a biochemical study of CpLDH1 in comparison with that of CpMalDH1 was necessary for the confirmation of their true biochemical functions. In particular, we were interested in monitoring whether putative CpLDH1 is able to display expected LDH activity with a Gly instead a Gln (most of LDH) at the discriminating 102 amino acid position. Such a functional characterization would also provide critical information for the future analysis on the drug actions against these two C. parvum enzymes.
FIG. 2. Alignment of L-malate dehydrogenase (MalDH) and L-lactate dehydrogenase (LDH) amino acid sequences from the Apicomplexa and other representative species at the N-terminal extensions and catalytic site. The ClustalW alignment was performed using the MacVector version 7.1.1 program. The active site loop is underlined, and the key amino acid denoting substrate specificity is boxed. Dashes represent gaps introduced to optimize alignment. Asterisks indicate residues that are conserved among all species. Positions containing the same amino acids as from CpMalDH1 or CpLDH1 were highlighted in gray
Our analysis confirmed that the substrates for CpMalDH1 and CpLDH1 are oxaloacetate and pyruvate, respectively. The Michaelis constants (Km) for MBP-CpMalDH1 were 510 μM for oxaloacetate and 20 μM for NADH, and those values for MBP-CpLDH1 were similarly at 500 μM for NADH and pyruvate for 20 μM. Specific activity (Vmax) for MBP-CpMalDH1 to reduce oxaloacetate was 36.0 U/mg (1 U = μmol/min). Based on the observation that mainly dimmer was present in the reaction, we determined that the Kcat with oxaloacetate was 5,090 min-1 and Kcat/Km was 0.1 x 108 min-1 M-1 for MBP-CpMalDH1. Similarly, the Vmax value for MBP-CpLDH1 was 23.0 U/mg, whereas the Kcat and Kcat/Km with pyruvate were 1,630 min-1 and 0.32 x 108 min-1 M-1, respectively.
The enzyme kinetic data have been previously determined for many organisms, and their specific activities range from 150 to 880 U/min. In the case of LDH-like MalDHs, Km values have shown to range from 4 to 24 μM for NAD(P) and from 22 to 85 μM for oxaloacetate (Ohshima and Sakuraba 1986; Hartl et al. 1987; Wynne et al. 1996; Langelandsvik et al. 1997; Madern et al. 2000). The recombinant MBP-CpMalDH1 protein had a similar Km value and a lower Kcat/Km for OAA than other native enzymes from eubacterial (including those from -proteobacteria) and archaebacterial species. The MBP-CpLDH1 catalytic efficiency was also lower when compared with the LDHs from another apicomplexan, T. gondi (Dando et al. 2001).
Crystallographic analyses of various MalDHs and LDHs in the presence of substrates or coenzyme analogs have shown that some structural reorganizations take place during catalysis (Gerstein and Chothia 1991; Iwata et al. 1994; Chapman et al. 1999). In comparison with free enzymes, the extreme low enzymatic efficiency of recombinant C. parvum enzymes could be largely attributed to the presence of an MBP tag. The 40-kDa MBP tag appeared to have little effect on the catalytic sites of CpMalDH1 and CpLDH1, as indicated by their Km values, but it likely made the parasite fusion proteins less flexible (and therefore less efficient), thus affecting their oligomeric states. Nevertheless, our data clearly confirmed the function of CpMalDH1 and CpLDH1 proteins. In addition, both parasite enzymes seemed to be highly substrate specific, because no enzymatic activity was detectable when oxaloacetate or pyruvate was used as substrate for recombinant CpLDH1 or CpMalDH1 proteins, respectively (data not shown).
Plasmodium LDH has been demonstrated to be one of the drug targets for the antimalarial compound chloroquine (Menting et al. 1997; Read et al. 1999), although the major antimalarial activity of this quinoline is caused by the inhibition of haematin polymerization (Dorn et al. 1998). However, both recombinant CpLDH1 and CpMalDH1 proteins displayed no sensitivity at all to this drug (data not shown). Chloroquine was found to bind at the NADH-binding site and acts as a weak inhibitor of P. falciparum LDH (Read et al. 1999; Surolia 2000). Therefore, the insensitivity of C. parvum enzymes to this compound is likely attributed to the structural difference at the NADH-binding sites of MalDH/LDH enzymes between these two parasites. However, the divergence of apicomplexan MalDH/LDH enzymes from those of humans and animals still supports the notion that these enzymes may be explored as rational drug targets for other classes of compounds against apicomplexan-based diseases.
Phylogenetic Reconstructions of Apicomplexan MalDH and LDH Enzymes
Earlier phylogenetic reconstructions showed that apicomplexan MalDH/LDH enzymes formed a monophyletic group that was closely related to -proteobacterial LDH-like MalDHs (Golding and Dean 1998; Madern 2002; Zhu and Keithly 2002). However, only CpMalDH1 had been previously available, which was placed as a sister to CpLDH1, which implied a possible gene duplication evolution between these two C. parvum enzymes (Zhu and Keithly 2002). The phylogenetic position and evolutionary origin of other apicomplexan MalDHs remained unknown because of the lack of sequence data. Recently, several completed and ongoing apicomplexan genome projects have uncovered a number of MalDH genes from various species, including P. falciparum, P. knowlesi, T. gondii, and E. tenella, thus permitting a more detailed phylogenetic analysis of these enzymes.
One interesting observation from these genome projects was that P. falciparum and C. parvum possessed only a single type of MalDH gene. Such a fact could also be true to other apicomplexans, although complete sequencing of other apicomplexan species, including T. gondii and E. tenella, is needed to make firm conclusions. Sequence analysis indicated that all known apicomplexan MalDH enzymes were cytosolic based on the lack of N-terminal targeting sequences in these enzymes (fig. 2). The lack of mitochondrial MalDH in C. parvum agrees with the absence of Krebs' cycle in this parasite, which is supported by early biochemical data (Entrala and Mascaro 1997) and current data mining of the nearly completed genome sequence (Abrahamsen et al., unpublished data). Although C. parvum lacks a morphologically recognizable mitochondrion and a Krebs' pathway, recent data suggest that this parasite may possess a remnant organelle of mitochondrion origin on the basis of the localization of a mitochondrial heat shock protein (hsp60) to a double membrane bounded structure in sporozoites (Riordan et al. 2003). On the other hand, most other apicomplexans (i.e., Plasmodium, Toxoplasma, and Eimeria) possess typical mitochondria. It is yet unclear how these species complete their Krebs' cycle without a mitochondrial MalDH, although it has been speculated that a malate-quinone oxidoreductase could replace MalDH in the TCA cycle (Gardner et al. 2002). It is also unknown whether apicomplexans lost their mitochondrial MalDHs shortly after their evolutionary separation from other eukaryotes or simply never acquired mitochondrion-specific dimeric MalDH genes (if mitochondrial MalDHs in other organisms were acquired from a -proteobacterium after the Apicomplexa separated from other eukaryotes as indicated by various phylogenetic reconstructions).
Our phylogenetic reconstructions using ML and BI methods again supported a monophylogic relationship of all known apicomplexan LDH-like MalDHs and LDH enzymes (see close up in figure 3). All phylogenetic trees displayed almost identical topology and major clades were robustly supported by the ML bootstrapping, quartet-puzzling, and BI posterior probability values. Within the LDH-like MalDHs, the monophyletic relationship of apicomplexan MalDHs and LDH enzymes clearly differs from the paraphyletic relationship among the majority of prokaryotic and eukaryotic enzymes that are widespread throughout the two other groups (fig. 3A). If apicomplexans had possessed MalDH/LDH genes sharing a common ancestor with those of other organisms, these ancestral MalDH/LDH genes were probably lost before or shortly after the divergence of the Apicomplexa from the other eukaryotes.
FIG. 3. Phylogenetic relationships of MalDH and LDH enzymes. (A) Diagram of three major subgroups: dimeric MalDHs, LDHs, and LDH-like MalDHs. Nodes representing two separate ancestral gene duplications splitting dimeric MalDHs and the rest of homologs, as well as LDHs and LDH-like MalDHs, are labeled with "1" and "2," respectively. (B) Detailed close-up of the phylogeny of apicomplexan and –proteobacterial LDH-like MalDHs and LDHs. The best maximum-likelihood (ML) tree (-ln L = 7542.74) shown here was generated by ProML program from 278 amino acids of 25 LDH and MalDH sequences using a JTT model (JTT–f + + Inv.) of amino acid substitutions. Amino acid frequencies (f), fraction of invariance (Inv.) and 4 rate categories of gamma distribution () were estimated from the data set based on an neighbor-joining (NJ) topology using Tree-Puzzle version 5.0 program. Statistical supporting values indicated at branch nodes were calculated by ML bootstrapping analysis from 100 replicates and by posterior probability analysis of 4,500 best trees generated by Bayesian inference (BI) method. Nodes containing two independent gene duplication events that occurred to the ancestral apicomplexan and to C. parvum are numbered with "3" and "4," respectively. Asterisks indicate sequences that were retrieved from various apicomplexan genome-sequencing projects as raw sequence data (see Materials and Methods for details)
Within the Apicomplexa clade, all LDH-like MalDHs were placed at the base, whereas all LDHs (except the CpLDH1) emerged later as a monophyletic group. Such a tree topology clearly suggested that the majority of apicomplexan LDHs evolved from an ancestral LDH-like MalDH by a gene duplication event before the expansion of apicomplexan species. This event likely occurred after the Apicomplexa had diverged from other eukaryotes because the apicomplexan LDH-like MalDH/LDH clade contained no homologs from any other major taxonomic groups. However, it remains unclear whether this event occurred before or after the apicomplexans separated from other aveolates, and currently there are no MalDH/LDH sequences available from ciliates and dinoflagellates to resolve this issue.
Another striking fact was that CpLDH1 separated from all other apicomplexan LDHs and was placed as a sister to the CpMalDH1 within the LDH-like MalDH group. This observation indicated that, unlike other apicomplexan LDHs, CpLDH1 and CpMalDH1 most likely evolved from a common ancestor by a very recent event of gene duplication after C. parvum separated from other apicomplexans. This event was apparently independent of the earlier gene duplication, coupled with a functional conversion, that gave rise to the majority of apicomplexan LDHs. If Cryptosporidium had possessed an LDH gene that shared a common ancestor with other apicomplexan LDHs before, this old LDH gene was apparently lost and replaced by the current CpLDH1 gene. The evolution of CpLDH1 from duplicated CpMalDH1 is also supported by the fact that these genes are tandem repeats in the C. parvum genome (in chromosome VII) (Abrahamsen et al., unpublished data) and, unlike other apicomplexan LDHs, CpLDH1 does not possess a 5-aa insertion at its active site loop (all apicomplexan LDH-like MalDHs lack such an insertion, too) (fig. 2).
It has been thought for a long time that MalDHs and LDHs separated into two major lineages that contained tetrameric LDH and dimeric MalDH, respectively. This point of view was challenged by the sequence, structural, and biochemical analyses on the first tetrameric MalDH (Cendrin et al. 1993; Madern et al. 2000; Richard et al. 2000). Recent phylogenetic analysis has established that three groups exist (Madern 2002). In particular, tetrameric MalDH evolved to LDH-like enzymes as indicated by their close relationships with the majority of LDH enzymes. Present LDHs and LDH-like MalDHs are the result of a homologous ancestral gene duplication. MalDH enzymes that are members of the LDH-like group are very divergent (not orthologous) from enzymes that belong to the well-described cytosolic and mitochondrial dimeric MalDHs. The key to understanding the general MalDH and LDH gene evolution is given by the location of two kinds of mutations that have been selected on duplicated ancestral genes to give third groups. The first type of mutation concerns those accumulated at the interface between dimers of dimers that make tetramer assemblies. This can be determined using comparaison at the structural level. Three-dimensional (3D) superimposition between LDH and LDH-like MalDH, LDH and dimeric (mitochondrial/cytosolic) MalDH, and dimeric MalDH and LDH-like MalDH explains the various oligomeric states observed within the whole family (Madern et al., unpublished data). The well-described second type of mutation concerns a single amino acid at the position 102 in the catalytic site that is important in determining the substrate specificity of these enzymes (Wilks et al. 1988; Nicholls et al. 1992; Cendrin et al. 1993).
Despite the fact that only a single amino acid at the active site loop is critical to the substrate specificity of these enzymes, functional changes between MalDHs and LDHs have rarely occurred among major taxonomic groups studied so far, suggesting that nature has discovered a mechanism to prevent, at least most of the time, the conversion between MalDHs and LDHs. Artificially induced mutations at position 102 of various recombinant enzymes have shown that a true functional change mainly depends of the oligomeric state (Madern 2002). With the tetrameric folds, a true functional conversion (from lactate to malate dehydrogenase or vice versa) can be observed with Arg-102 LDH and Gln-102 LDH-like MalDH. In contrast, the dimeric fold still favors malate dehydrogenase activity and not lactate dehydrogenase activity in Gln-102 MalDHs. In this case, a true functional conversion may not be achieved solely by the removal of the charge alone from the amino acid at position 102, and additional mutations among other residues are required, as illustrated by the E. coli MalDH redesign into phenyllactate dehydrogenase (Wright, Kish, and Viola 2000). In living organisms that require both LDH and MalDH activities to sustain their cellular functions, they usually possess a typical LDH, whereas the MalDH can be either a dimeric MalDH (cytosolic or mitochondrial subgroup) or a tetrameric LDH-like MalDH. The former appears to occur more frequently than the latter. Natural selection of enzymes in favor to the dimeric MalDHs with more efficient and stringent function than LDH-like MalDHs was therefore a very likely means to prevent the selection of new LDHs from MalDH mutations.
Previously, the evolution of LDH from cytosolic MalDH (rooted within the dimeric group) was reported for T. vaginalis (Wu et al. 1999). This functional conversion has lead to an LDH that has conserved a high MalDH activity. In this study, we have observed that apicomplexan LDH enzymes were likely converted from a duplicated LDH-like MalDH gene originally acquired from an -proteobacterium (see node labeled "3" in figure 3B). This gene duplication led to the creation of a new type of LDH and was independent of the ancestral separation between LDH and LDH-like MalDHs (see node labeled "2" in figure 3A). In fact, these apicomplexan LDHs might be considered as LDH-like MalDH–resembling enzymes. Independently from this functional conversion of apicomplexan LDHs from an -proteobacterial MalDH, CpLDH1 and CpMalDH1 apparently evolved from the same ancestor (likely an MalDH1 gene) by a very recent event of gene duplication (see node labeled "4" in figure 3B).
Similar cases of functional conversion within the clade of dimeric MalDHs were described in kinetoplastids (Cazzulo Franke et al. 1999; Uttaro et al. 2000). In this clade, MalDH from Trypanosoma cruzi and Phytomonas have independently evolved twice to give new enzymes with a broader range of substrate specificity. In the case of Phytomonas the recent gene duplication of the glycosomal MalDH has lead to a 2-hydroxyacid dehydrogenase able to recognize phenyl pyruvate, 2-oxocaproate, and 2-oxoisocaproate in addition to oxaloacetate (Uttaro and Opperdoes 1997). All the functionnal conversions mentioned here suggest that, within the MalDHs (LDH-like and dimeric), the substrate recognition mechanism between dicarboxylic and monocarboxylic acid was bypassed at least four times during evolution. These events reflect the powerful organism plasticity in response to changes in their environments.
The different origin of CpLDH1 from other apicomplexan LDHs also provides additional evidence on the molecular and evolutionary divergence of C. parvum from other apicomplexans. Indeed, although conventional taxonomy has placed the Cryptosporidium genus within the Class Coccidea. mainly based on strong morphological evidence, several early phylogenetic reconstructions have clearly placed this genus at the base of the Apicomplexa (i.e., monophyletic to Coccidia + Haematozoa) (Morrison and Ellis 1997; Zhu, Keithly, and Philippe 2000) or as a sister to the gregarines (Carreno, Martin, and Barta 1999). Unlike other apicomplexans, C. parvum apparently lacks plastid and mitochondrial genomes, based on experimental data (Zhu, Marchewka, and Keithly 2000) or data mining the nearly complete genome project. All these observations suggest that Cryptosporidium has a unique evolutionary history that differs from other apicomplexans at both organismal and metabolic levels.
Acknowledgements
This research was supported in part by grants from the National Institute of Allergy and Infectious Diseases (NIAID) at the National Institutes of Health (NIH), U.S. Department of Human Health Services (DHHS) (R01 AI44594 and R21 AI055278 to G.Z. and U01 AI046397 to M.S.A.) and by a grant from the European Economic Community (EEC): "European network for biological deuteration HPRI CT 2001 50035" to D.M. Preliminary sequence data for Toxoplasma gondii malate dehydrogenase (MalDH) was obtained from The Institute for Genomic Research Web site at http://www.tigr.org. Sequencing of T. gondii genome was funded by NIAID. Preliminary sequence data for Eimeria tenella MalDH were produced by the E. tenella genome project at the Sanger Institute and can be obtained from ftp://ftp.sanger.ac.uk/pub/databases/E.tenella/, which was funded by the U.K. Biotechnology and Biological Science Research Council (BBSRC).
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Department of Veterinary Pathobiology, Texas A&M University, College Station
Department of Veterinary Pathobiology, University of Minnesota, St. Paul
E-mail: g3hu@cvm.tamu.edu.
Abstract
We have expressed the L-lactate dehydrogenase (LDH) and L-malate dehydrogenase (malDH) genes from the apicomplexan Cryptosporidium parvum (CpLDH1 and CpMalDH1) as maltose-binding protein (MBP) fusion proteins in Escherichia coli. The substrate specificities, enzymatic kinetics, and oligomeric states of these two parasite enzymes have been characterized. By taking advantage of recently completed and ongoing apicomplexan genome sequencing projects, we identified additional MalDH genes from Plasmodium spp., Toxoplasma gondii, and Eimeria tenella that were previously unavailable. All apicomplexan MalDHs appeared to be cytosolic and no organellar homologs were identified from the completely sequenced P. falciparum genome and other ongoing apicomplexan genome-sequencing projects. Using these expanded apicomplexan LDH and MalDH sequence databases, we reexamined their phylogenetic relationships and reconfirmed their relationship to -proteobacterial MalDHs. All LDH and MalDH enzymes from apicomplexans were monophyletic within the LDH-like MalDH group (i.e., MalDH resembling LDH) as a sister to -proteobacterial MalDHs. All apicomplexan LDHs, with the exception of CpLDH1, formed a separate clade from their MalDH counterparts, indicating that these LDHs were evolved from an ancestral apicomplexan MalDH by a gene duplication coupled with functional conversion before the expansion of apicomplexans. Finally, CpLDH1 was consistently placed together with CpMalDH1 within the apicomplexan MalDH cluster, confirming an early working hypothesis that CpLDH1 was probably evolved from the same ancestor of CpMalDH1 by a very recent gene duplication that occurred after C. parvum diverged from other apicomplexans.
Key Words: Apicomplexa ? Cryptosporidium parvum ? malate dehydrogenase ? lactate dehydrogenase ? phylogeny ? evolution ? gene duplication
Introduction
L-malate dehydrogenase (L-MalDH) and L-lactate dehydrogenase (L-LDH) belong to the superfamily of 2-ketoacid:NAD(P)–dependent dehydrogenases that catalyze the reversible conversion of 2-hydroxyacids to the corresponding 2-ketoacids. MalDHs and LDHs (all MalDHs and LDHs herein refer to L-MalDH and L-LDHs, respectively) have been purified and characterized from a wide variety of organisms. MalDHs were found to be widespread over the three domains of life, whereas LDHs are solely present in Eukarya and Bacteria. Recent phylogenetic reconstructions have shown that two ancestral paralogous gene duplications explain the main functional distribution and evolution of MalDH and LDH (Madern 2002). These duplications have lead to the existence of three major clades of enzymes that differ strongly in their structural and biochemical features (Madern 2002). The first clade includes the two well-described subgroups of mitochondrial and cytosolic dimeric MalDHs (Roger, Morrison, and Sogin 1999). The others are the LDH-like MalDHs (i.e., MalDH resembling LDH) and LDHs. LDH-like MalDHs and LDHs have a similar tetrameric architecture based on crystallographic analysis (Richard et al. 2000; Lee et al. 2001; Dalhus et al. 2002). Therefore, in contrast to previous scenarios of molecular evolution between MalDHs and LDHs (described without any knowledge of the LDH-like MalDHs), Madern (2002) proposed that LDHs were not the result of a primary ancestral duplication from MalDHs (in the dimeric state), but were very likely selected from an ancient gene duplication with LDH-like MalDHs.
According to the general numbering scheme for LDH and MalDH enzymes (Eventoff et al. 1977), residues at position 102 are the most important in determining substrate specificity (Wilks et al. 1988; Nicholls et al. 1992; Cendrin et al. 1993), which is typically a charged arginine in MalDHs and an uncharged residue (mostly glutamine) in LDHs, respectively. Mutagenesis experiments have clearly demonstrated that single amino acid change from glutamine to arginine at the position 102 might change LDH to MalDH (Wilks et al. 1988; Goward and Nicholls 1994; Golding and Dean 1998), although functional conversion from MalDH to LDH has been much less successful by replacing arginine with an uncharged residue (Cendrin et al. 1993; Boernke et al. 1995; Wu et al. 1999). However, besides the two ancestral gene duplications mentioned above, recent functional changes between MalDH and LDH appeared to rarely occur in the nature. There has been no evidence that the LDH lineage contains any MalDH enzymes. On the other hand, only a few types of LDHs were intermixed within the various MalDH groups. For example, trichomonad LDHs were found linked with the cytosolic dimeric MalDH (Cazzulo Franke et al. 1999), whereas apicomplexan LDHs were rooted with LDH-like MalDH (Wu et al. 1999; Zhu and Keithly 2002). The trichomonad LDHs probably evolved from their cytosolic dimeric MalDH in recent events of gene duplication. Apicomplexan LDH and MalDH enzymes were found to be monophyletic to the -proteobacterial LDH-like MalDHs, suggesting that these parasite enzymes might be acquired from an -proteobacterium by a lateral gene transfer (LGT) event or by retaining the bacterial LDH-like MalDHs during the primary endosymbiosis that gave rise to mitochondria. In agreement with these individual observations, the general molecular evolution pathway of MalDHs and LDHs seems to be polarized from high to low stringency of substrate recognition, leading to functional changes of MalDHs towards LDHs without any evidence for the reverse phenomenon (Madern 2002).
Among apicomplexans, early studies have shown that Toxoplasma gondii possess two LDH genes that are differentially expressed during their complex life cycle (Yang and Parmley 1997) Only one LDH gene from Plasmodium falciparum was cloned and intensively studied (Simmons et al. 1985), although, recently, genome projects revealed the presence of another putative, but a highly divergent, LDH gene in this parasite (i.e., GenBank accession number CAD52400). Analysis of the completely sequenced P. falciparum genome and the ongoing T. gondii genome project supports the belief that both organisms only have these two LDH genes. Furthermore, a single MalDH gene encoding a cytosolic enzyme was recently identified from each of these two apicomplexan genomes. In contrast, only one each of the putative MalDH and LDH genes from Cryptosporidium parvum has been previously reported, and no additional MalDH/LDH genes could be found within its completed genome sequence. In this study, we expressed the both C. parvum MalDH and LDH in Escherichia coli as maltose-binding protein (MBP) fusion proteins to correctly assess their respective enzymatic function and oligomeric states in solutions. With the expanded MalDH and LDH sequences from both apicomplexans and -proteobacteria, new phylogenetic reconstructions have reconfirmed our early observations that these enzymes are linked to the LDH-like MalDHs (Madern 2002; Zhu and Keithly 2002). They also revealed that all apicomplexan LDH-like MalDHs and LDH enzymes were monophyletic, which suggested that most apicomplexan LDH-like MalDHs and LDHs evolved from a common ancestor by a gene duplication before the evolutionary expansion of apicomplexans. More interestingly, C. parvum LDH gene appeared to evolve from its cytosolic partner, LDH-like MalDH (independently from those of other apicomplexan LDHs), by a very recent gene duplication that occurred after this organism diverged from other apicomplexans.
Materials and Methods
Expression and Biochemical Characterization of CpMalDH1 and CpLDH1 Enzymes
Two genes encoding putative MalDH and LDH enzymes were previously identified from C. parvum, which were designated to CpMalDH1 and CpLDH1, respectively (Zhu and Keithly 2002). Their nucleotide sequences were deposited in GenBank under the accession numbers AF274310 (CpLDH1) and AY334274 (CpMalDH1). Their initial annotations were based on the key amino acid at the active site loop in which CpMalDH1 possessed an arginine, and CpLDH1 had an uncharged glycine that differed from the glutamine found in the majority of LDHs. Data mining the nearly completely sequenced C. parvum genome indicated that CpMalDH1 and CpLDH1 were the only 2-ketoacid dehydrogenase homologs possessed by this parasite.
The entire open reading frames (ORFs) of the CpMalDH1 and CpLDH1 genes were amplified by PCR using high-fidelity AccuTaq DNA polymerase (Sigma Chemical Co., St Louis, Mo.) for cloning into a pMAL-c2x vector (New England Biolabs, Beverly, Mass.) at the EcoRI restriction site. Because the cpMalDH gene contained internal EcoRI sites, a strategy to avoid post-PCR digestion of amplicons with EcoRI was employed (fig. 1): first, oligonucleotide linkers "aattc" (long type) or "c" (short type) was added to each of the sense and antisense primers; second, the long type sense primers were paired only with the short antisense ones (and vice versa) in PCR to produce two types of amplicons for each of the two genes; third, all PCR products were phosphorylated with T7 DNA kinase, and the kinase was then removed using a DNA digestion cleanup kit (Qiagen, Valencia, Calif); and fourth, the same amounts of the separately amplified PCR products for each gene were mixed together and denatured by heating at 100°C for 10 min and slowly cooling down to room temperature within a heat block. Theoretically, 25% of the reannealed amplicons would contain the phosphorylated 5'-aatt overhanging at both ends, which could be subsequently ligated into pMAL-c2x vector (fig. 1).
FIG. 1. Amplification of CpMalDH1 and CpLDH1 open reading frames (ORFs). Because CpMalDH1 ORF contained an internal EcoRI restriction site, a strategy to avoid post-PCR digestion of EcoRI by directly creating 5'-aatt overhanging ends was employed during the amplification and engineering of CpMDH1 and CpLDH1 genes into the pMAL-c2x at the EcoRI restriction site. Letters in lowercase represent artificially added nucleotide linkers
After ligation and transformation into E. coli competent cells, bacterial clones were subjected to direct PCR screening to determine the orientation of their inserts. Plasmids containing correctly oriented CpMalDH1 or CpLDH1 inserts were sequenced to confirm their identities and transformed into an E. coli Rosetta strain (Novagen, Madison, Wis.) for the expression of MBP-fusion proteins. Bacterial transformants were allowed to grow at 37°C until OD590 reached 0.4 to 0.6, which was followed by the addition of 0.5 mM IPTG and subsequent growth at 20°C for 6 h. The bacteria were collected by centrifugation and disrupted by sonication at 4°C in a binding buffer containing PMSF, EDTA, and pepstatin. Recombinant CpMalDH1 and CpLDH1 proteins were purified from supernatants using an amylose resin-based affinity chromatography according to the manufacturer's instructions. In some cases, the fusion proteins were further purified using ion exchange chromatography (Mono-Q resin). A portion of the purified fusion proteins was subjected to factor Xa digestion to separate the MBP-tag and parasite proteins. The purity of fusion proteins was analyzed in 10% SDS-PAGE gels, and their biochemical properties were characterized as described below.
The enzymatic activity of recombinant MBP-CpMalDH1 or MBP-CpLDH1 was measured in 50 mM Tris HCl buffer (pH 8.0) containing 0.2 mM NADH and
0.3 mM oxaloacetate or pyruvate, respectively. NADH oxidation by both enzymes was monitored at 340 nm on a Beckman DU7400 spectrophotometer at 25°C. The Km and Vmax values of the fusion proteins were determined for NADH, pyruvate, and oxaloacetate, respectively. Using the same assays, we also tested whether CpLDH1 activity could be inhibited by chloroquine, a potent inhibitor of P. falciparum LDH (Read et al. 1999).
The oligomeric states of these two parasite fusion proteins were analyzed by a size exclusion chromatography (Sephacryl S200). Sedimentation velocity analysis was performed on the recombinant proteins. This method has been shown to be very efficient for determining unambiguously the oligomeric state of LDH-like MalDH (Madern et al. 2000). Experiments were performed on a Beckman XLA analytical ultracentrifuge, equipped with a UV scanning system, using a four hole AN-60 Ti rotor with double centerpieces of 1.20 cm path-length. In a typical experiment, 200 absorbance profiles for each sample were recorded at 42,000 rpm at 20°C. The wavelengths were chosen according to the characteristics of the samples. The scan profiles were analyzed with the SEDFIT program (Schuck 2000) using the continuous sedimentation (or mass) distribution option. The partial specific volume of MBP-CpLDH1 (2 = 0.7419) and MBP-CpMalDH1 (2 = 0.7439) were determined from their primary sequence. The coefficients, S20,W, were corrected for temperature and viscosity using tabulated viscosity and density . Theoretical S values were calculated using the predicRHDC software.
Phylogenetic Reconstructions of Apicomplexan MalDH and LDH Enzymes
The cloning and sequencing of CpMalDH1 and CpLDH1 enzymes, as well as those of P. falciparum and T. gondii LDHs were previously described (Zhu and Keithly 2002). However, only one MalDH (i.e., CpMalDH1) sequence was previously available (Zhu and Keithly 2002). Therefore, the phylogenetic relationships of apicomplexan MalDHs and LDHs were not fully studied before. More recently, with the completion of P. falciparum (Haematozoa) genome (http://www.plasmodb.org) and the rapid progress of the ongoing T. gondii (cyst-forming Coccidia) (http://www.tigr.org/tdb/e2k1/tga1) and Eimeria tenella (intestinal Coccidia) (http://www.sanger.ac.uk/Projects/E_tenella) genome-sequencing projects, we were able to retrieve several apicomplexan MalDH genes from their corresponding databases. Because apicomplexan MalDH/LDH enzymes have shown to be evolutionarily related to the -proteobacterial LDH-like MalDHs (Zhu and Keithly 2002), a number of MalDH genes from this group of bacteria were also retrieved from the GenBank and used as an outgroup in the phylogenetic analysis. In total, seven apicomplexan LDH, five apicomplexan MalDH, and 13 -proteobacterial MalDH enzymes were included in the phylogenetic reconstructions.
All MalDH/LDH protein sequences were conceptually translated from their corresponding genes and aligned together using the MacVector version 7.1.1 program (Acceryl, Inc.). Aligned sequences were visually inspected to correct apparent mistakes. Finally, 278 unambiguously aligned amino acid positions were used in the analysis. The Tree-Puzzle version 5.0 program (Strimmer and von Haeseler 1996) was first used to generate approximate quartet likelihood trees with 10,000 puzzling steps. Parameters were calculated from the topology of a neighbor-joining tree and the amino acid frequencies (f) were estimated from data sets using a JTT model of amino acid substitution with the consideration of rate heterogeneity; that is, the fraction of invariance and four-rate gamma distributions (JTT–f + + Inv.). Parameters established from puzzling analysis were then applied to a true maximum-likelihood (ML) analysis using a ProML program in the PHYLIP version 3.6a2 package (Felsenstein 1993) with sequence input order randomized and global rearrangements enabled during the tree search. Similarly, ML bootstrapping analysis was performed using the ProML program from 100 replicates generated by the SeqBoot program. In addition, phylogenetic trees were also reconstructed with the JTT model of amino acid substitutions and the same rates of heterogeneity (JTT–f + + Inv.) using a Bayesian inference (BI) method (Huelsenbeck and Ronquist 2001). A total of 500,000 generations of searches were performed with four chains simultaneously running. Stable ML values were quickly reached before 10,000 generations of searches, indicating that the Markov chain Monte Carlo (MCMC) application was allowed to run for sufficient generations. Posterior probabilities at tree nodes were obtained by calculating the consensus tree from the best 4,500 BI trees using a 50% majority ruling method.
Results and Discussions
Characterization of CpMalDH1 and CpLDH1 Oligomeric States
Our phylogenetic analysis (see below) and previous reconstructions have shown that apicomplexan MalDHs and LDHs are rooted within the group of LDH-like MalDHs (Madern 2002; Zhu and Keithly 2002). Crystallographic structures of three LDH-like MalDH have revealed their tetrameric organization (dimer of dimers) (Richard et al. 2000; Lee et al. 2001; Dalhus et al. 2002). According to this, our aim was to determine more precisely the respective oligomeric state of CpMalDH1 or CpLDH1 (both expected to be tetrameric). Unfortunately, we encountered some technical difficulties to efficiently separate the CpMalDH1 or CpLDH1 portion from the MBP-tag by digesting fusion proteins with factor Xa. We decided therefore to record data using pure MBP-fusion proteins.
In a first attempt, we analyzed the oligomeric state of both MBP-fusions by size exclusion chromatography using a well-calibrated column. In contrast to our expectations, recombinant active MBP-fusion proteins were recorded with an elution volume higher than that estimated for tetrameric MBP-fusions, indicating they were not tetrameric. To get more accurate data, we have recorded the enzyme properties using analytical centrifugation (AUC).
Pure tetrameric MBP-CpMalDH1 and MBP-CpLDH1 would migrate in an AUC experiment as single species with theoritical sedimentation coefficient of S20,W = 12.5 S corresponding to spherical proteins of 316 kDa. In contrast to our expectation, a sedimentation velocity analysis of MBP-CpMalDH1 protein identified two main species with lower apparent sedimentation coefficiencies. The data (not shown) were well fitted with a similar amount of slightly elongated dimeric and monomeric state of MBP-CpMalDH1 with S20,W of 6.2 S and 3.9 S, respectively. A tiny fraction of bigger species (< 2%) was also observed that could have been caused by a nonspecific aggregation rather than by a very small amount of tetrameric molecules. The two major species displayed apparent molecular weights at 73 and 156 kDa, respectively, which were close to the theoretical values of a monomer (76 kDa) and a dimer (153 kDa) for the MBP-CpMalDH1 fusion. Analysis of the structural information available for LDH-like MalDHs shows that a distortion of the N-terminus might propagate toward the region that controls the association between monomers. In our case, the data suggest that MBP-tag fused at the N-terminal part of CpMalDH has very likely induced some structural reorganizations (because of steric hindrance) that has drastically modified the folding and association pathway, allowing it to trap intermediates that exists during this process. Using a recombinant enzyme from Haloarcula marismortui, dimeric intermediate species that assemble to make a tetrameric LDH-like MalDH have been demonstrated to be active (Madern et al. 2000). According to this observation, the purified MBP-CpMalDH1 that contains dimeric species would be satisfactory to determine enzymatic properties.
Similarly, two species of MBP-CpLDH1 protein (instead of a single species of 317 kDa with S20,W = 12.5 S) were also present in solution with apparent S20,W of 6.0 S and 4.2 S, respectively. Their molecular weights, determined by the mass distribution option of Sedfit, were 77 and 134 kDa, respectively, which agreed with calculated molecular weights of MBP-CpLDH1 (i.e., monomer Mr = 77 kDa and a dimer Mr = 153 kDa). As discussed previously, the same analysis and conclusion holds for MBP-CpLDH1. All of these observations suggested a monomer-dimer equilibrium for both parasite fusion proteins in solution.
Enzymatic Properties of CpMalDH1 and CpLDH1
It has been shown that P. falciparum and T. gondii LDHs differ from other organisms (including their hosts) by possessing a 5-aa insertion at their catalytic sites, which could imply a unique mode of action of these enzymes. The 5-aa insertion is also present in recently cloned E. tenella, E. acervulina, and E. maxima LDHs (fig. 2). However, this unique insertion is not present in CpLDH1 or any of the apicomplexan MalDHs. Therefore, a biochemical study of CpLDH1 in comparison with that of CpMalDH1 was necessary for the confirmation of their true biochemical functions. In particular, we were interested in monitoring whether putative CpLDH1 is able to display expected LDH activity with a Gly instead a Gln (most of LDH) at the discriminating 102 amino acid position. Such a functional characterization would also provide critical information for the future analysis on the drug actions against these two C. parvum enzymes.
FIG. 2. Alignment of L-malate dehydrogenase (MalDH) and L-lactate dehydrogenase (LDH) amino acid sequences from the Apicomplexa and other representative species at the N-terminal extensions and catalytic site. The ClustalW alignment was performed using the MacVector version 7.1.1 program. The active site loop is underlined, and the key amino acid denoting substrate specificity is boxed. Dashes represent gaps introduced to optimize alignment. Asterisks indicate residues that are conserved among all species. Positions containing the same amino acids as from CpMalDH1 or CpLDH1 were highlighted in gray
Our analysis confirmed that the substrates for CpMalDH1 and CpLDH1 are oxaloacetate and pyruvate, respectively. The Michaelis constants (Km) for MBP-CpMalDH1 were 510 μM for oxaloacetate and 20 μM for NADH, and those values for MBP-CpLDH1 were similarly at 500 μM for NADH and pyruvate for 20 μM. Specific activity (Vmax) for MBP-CpMalDH1 to reduce oxaloacetate was 36.0 U/mg (1 U = μmol/min). Based on the observation that mainly dimmer was present in the reaction, we determined that the Kcat with oxaloacetate was 5,090 min-1 and Kcat/Km was 0.1 x 108 min-1 M-1 for MBP-CpMalDH1. Similarly, the Vmax value for MBP-CpLDH1 was 23.0 U/mg, whereas the Kcat and Kcat/Km with pyruvate were 1,630 min-1 and 0.32 x 108 min-1 M-1, respectively.
The enzyme kinetic data have been previously determined for many organisms, and their specific activities range from 150 to 880 U/min. In the case of LDH-like MalDHs, Km values have shown to range from 4 to 24 μM for NAD(P) and from 22 to 85 μM for oxaloacetate (Ohshima and Sakuraba 1986; Hartl et al. 1987; Wynne et al. 1996; Langelandsvik et al. 1997; Madern et al. 2000). The recombinant MBP-CpMalDH1 protein had a similar Km value and a lower Kcat/Km for OAA than other native enzymes from eubacterial (including those from -proteobacteria) and archaebacterial species. The MBP-CpLDH1 catalytic efficiency was also lower when compared with the LDHs from another apicomplexan, T. gondi (Dando et al. 2001).
Crystallographic analyses of various MalDHs and LDHs in the presence of substrates or coenzyme analogs have shown that some structural reorganizations take place during catalysis (Gerstein and Chothia 1991; Iwata et al. 1994; Chapman et al. 1999). In comparison with free enzymes, the extreme low enzymatic efficiency of recombinant C. parvum enzymes could be largely attributed to the presence of an MBP tag. The 40-kDa MBP tag appeared to have little effect on the catalytic sites of CpMalDH1 and CpLDH1, as indicated by their Km values, but it likely made the parasite fusion proteins less flexible (and therefore less efficient), thus affecting their oligomeric states. Nevertheless, our data clearly confirmed the function of CpMalDH1 and CpLDH1 proteins. In addition, both parasite enzymes seemed to be highly substrate specific, because no enzymatic activity was detectable when oxaloacetate or pyruvate was used as substrate for recombinant CpLDH1 or CpMalDH1 proteins, respectively (data not shown).
Plasmodium LDH has been demonstrated to be one of the drug targets for the antimalarial compound chloroquine (Menting et al. 1997; Read et al. 1999), although the major antimalarial activity of this quinoline is caused by the inhibition of haematin polymerization (Dorn et al. 1998). However, both recombinant CpLDH1 and CpMalDH1 proteins displayed no sensitivity at all to this drug (data not shown). Chloroquine was found to bind at the NADH-binding site and acts as a weak inhibitor of P. falciparum LDH (Read et al. 1999; Surolia 2000). Therefore, the insensitivity of C. parvum enzymes to this compound is likely attributed to the structural difference at the NADH-binding sites of MalDH/LDH enzymes between these two parasites. However, the divergence of apicomplexan MalDH/LDH enzymes from those of humans and animals still supports the notion that these enzymes may be explored as rational drug targets for other classes of compounds against apicomplexan-based diseases.
Phylogenetic Reconstructions of Apicomplexan MalDH and LDH Enzymes
Earlier phylogenetic reconstructions showed that apicomplexan MalDH/LDH enzymes formed a monophyletic group that was closely related to -proteobacterial LDH-like MalDHs (Golding and Dean 1998; Madern 2002; Zhu and Keithly 2002). However, only CpMalDH1 had been previously available, which was placed as a sister to CpLDH1, which implied a possible gene duplication evolution between these two C. parvum enzymes (Zhu and Keithly 2002). The phylogenetic position and evolutionary origin of other apicomplexan MalDHs remained unknown because of the lack of sequence data. Recently, several completed and ongoing apicomplexan genome projects have uncovered a number of MalDH genes from various species, including P. falciparum, P. knowlesi, T. gondii, and E. tenella, thus permitting a more detailed phylogenetic analysis of these enzymes.
One interesting observation from these genome projects was that P. falciparum and C. parvum possessed only a single type of MalDH gene. Such a fact could also be true to other apicomplexans, although complete sequencing of other apicomplexan species, including T. gondii and E. tenella, is needed to make firm conclusions. Sequence analysis indicated that all known apicomplexan MalDH enzymes were cytosolic based on the lack of N-terminal targeting sequences in these enzymes (fig. 2). The lack of mitochondrial MalDH in C. parvum agrees with the absence of Krebs' cycle in this parasite, which is supported by early biochemical data (Entrala and Mascaro 1997) and current data mining of the nearly completed genome sequence (Abrahamsen et al., unpublished data). Although C. parvum lacks a morphologically recognizable mitochondrion and a Krebs' pathway, recent data suggest that this parasite may possess a remnant organelle of mitochondrion origin on the basis of the localization of a mitochondrial heat shock protein (hsp60) to a double membrane bounded structure in sporozoites (Riordan et al. 2003). On the other hand, most other apicomplexans (i.e., Plasmodium, Toxoplasma, and Eimeria) possess typical mitochondria. It is yet unclear how these species complete their Krebs' cycle without a mitochondrial MalDH, although it has been speculated that a malate-quinone oxidoreductase could replace MalDH in the TCA cycle (Gardner et al. 2002). It is also unknown whether apicomplexans lost their mitochondrial MalDHs shortly after their evolutionary separation from other eukaryotes or simply never acquired mitochondrion-specific dimeric MalDH genes (if mitochondrial MalDHs in other organisms were acquired from a -proteobacterium after the Apicomplexa separated from other eukaryotes as indicated by various phylogenetic reconstructions).
Our phylogenetic reconstructions using ML and BI methods again supported a monophylogic relationship of all known apicomplexan LDH-like MalDHs and LDH enzymes (see close up in figure 3). All phylogenetic trees displayed almost identical topology and major clades were robustly supported by the ML bootstrapping, quartet-puzzling, and BI posterior probability values. Within the LDH-like MalDHs, the monophyletic relationship of apicomplexan MalDHs and LDH enzymes clearly differs from the paraphyletic relationship among the majority of prokaryotic and eukaryotic enzymes that are widespread throughout the two other groups (fig. 3A). If apicomplexans had possessed MalDH/LDH genes sharing a common ancestor with those of other organisms, these ancestral MalDH/LDH genes were probably lost before or shortly after the divergence of the Apicomplexa from the other eukaryotes.
FIG. 3. Phylogenetic relationships of MalDH and LDH enzymes. (A) Diagram of three major subgroups: dimeric MalDHs, LDHs, and LDH-like MalDHs. Nodes representing two separate ancestral gene duplications splitting dimeric MalDHs and the rest of homologs, as well as LDHs and LDH-like MalDHs, are labeled with "1" and "2," respectively. (B) Detailed close-up of the phylogeny of apicomplexan and –proteobacterial LDH-like MalDHs and LDHs. The best maximum-likelihood (ML) tree (-ln L = 7542.74) shown here was generated by ProML program from 278 amino acids of 25 LDH and MalDH sequences using a JTT model (JTT–f + + Inv.) of amino acid substitutions. Amino acid frequencies (f), fraction of invariance (Inv.) and 4 rate categories of gamma distribution () were estimated from the data set based on an neighbor-joining (NJ) topology using Tree-Puzzle version 5.0 program. Statistical supporting values indicated at branch nodes were calculated by ML bootstrapping analysis from 100 replicates and by posterior probability analysis of 4,500 best trees generated by Bayesian inference (BI) method. Nodes containing two independent gene duplication events that occurred to the ancestral apicomplexan and to C. parvum are numbered with "3" and "4," respectively. Asterisks indicate sequences that were retrieved from various apicomplexan genome-sequencing projects as raw sequence data (see Materials and Methods for details)
Within the Apicomplexa clade, all LDH-like MalDHs were placed at the base, whereas all LDHs (except the CpLDH1) emerged later as a monophyletic group. Such a tree topology clearly suggested that the majority of apicomplexan LDHs evolved from an ancestral LDH-like MalDH by a gene duplication event before the expansion of apicomplexan species. This event likely occurred after the Apicomplexa had diverged from other eukaryotes because the apicomplexan LDH-like MalDH/LDH clade contained no homologs from any other major taxonomic groups. However, it remains unclear whether this event occurred before or after the apicomplexans separated from other aveolates, and currently there are no MalDH/LDH sequences available from ciliates and dinoflagellates to resolve this issue.
Another striking fact was that CpLDH1 separated from all other apicomplexan LDHs and was placed as a sister to the CpMalDH1 within the LDH-like MalDH group. This observation indicated that, unlike other apicomplexan LDHs, CpLDH1 and CpMalDH1 most likely evolved from a common ancestor by a very recent event of gene duplication after C. parvum separated from other apicomplexans. This event was apparently independent of the earlier gene duplication, coupled with a functional conversion, that gave rise to the majority of apicomplexan LDHs. If Cryptosporidium had possessed an LDH gene that shared a common ancestor with other apicomplexan LDHs before, this old LDH gene was apparently lost and replaced by the current CpLDH1 gene. The evolution of CpLDH1 from duplicated CpMalDH1 is also supported by the fact that these genes are tandem repeats in the C. parvum genome (in chromosome VII) (Abrahamsen et al., unpublished data) and, unlike other apicomplexan LDHs, CpLDH1 does not possess a 5-aa insertion at its active site loop (all apicomplexan LDH-like MalDHs lack such an insertion, too) (fig. 2).
It has been thought for a long time that MalDHs and LDHs separated into two major lineages that contained tetrameric LDH and dimeric MalDH, respectively. This point of view was challenged by the sequence, structural, and biochemical analyses on the first tetrameric MalDH (Cendrin et al. 1993; Madern et al. 2000; Richard et al. 2000). Recent phylogenetic analysis has established that three groups exist (Madern 2002). In particular, tetrameric MalDH evolved to LDH-like enzymes as indicated by their close relationships with the majority of LDH enzymes. Present LDHs and LDH-like MalDHs are the result of a homologous ancestral gene duplication. MalDH enzymes that are members of the LDH-like group are very divergent (not orthologous) from enzymes that belong to the well-described cytosolic and mitochondrial dimeric MalDHs. The key to understanding the general MalDH and LDH gene evolution is given by the location of two kinds of mutations that have been selected on duplicated ancestral genes to give third groups. The first type of mutation concerns those accumulated at the interface between dimers of dimers that make tetramer assemblies. This can be determined using comparaison at the structural level. Three-dimensional (3D) superimposition between LDH and LDH-like MalDH, LDH and dimeric (mitochondrial/cytosolic) MalDH, and dimeric MalDH and LDH-like MalDH explains the various oligomeric states observed within the whole family (Madern et al., unpublished data). The well-described second type of mutation concerns a single amino acid at the position 102 in the catalytic site that is important in determining the substrate specificity of these enzymes (Wilks et al. 1988; Nicholls et al. 1992; Cendrin et al. 1993).
Despite the fact that only a single amino acid at the active site loop is critical to the substrate specificity of these enzymes, functional changes between MalDHs and LDHs have rarely occurred among major taxonomic groups studied so far, suggesting that nature has discovered a mechanism to prevent, at least most of the time, the conversion between MalDHs and LDHs. Artificially induced mutations at position 102 of various recombinant enzymes have shown that a true functional change mainly depends of the oligomeric state (Madern 2002). With the tetrameric folds, a true functional conversion (from lactate to malate dehydrogenase or vice versa) can be observed with Arg-102 LDH and Gln-102 LDH-like MalDH. In contrast, the dimeric fold still favors malate dehydrogenase activity and not lactate dehydrogenase activity in Gln-102 MalDHs. In this case, a true functional conversion may not be achieved solely by the removal of the charge alone from the amino acid at position 102, and additional mutations among other residues are required, as illustrated by the E. coli MalDH redesign into phenyllactate dehydrogenase (Wright, Kish, and Viola 2000). In living organisms that require both LDH and MalDH activities to sustain their cellular functions, they usually possess a typical LDH, whereas the MalDH can be either a dimeric MalDH (cytosolic or mitochondrial subgroup) or a tetrameric LDH-like MalDH. The former appears to occur more frequently than the latter. Natural selection of enzymes in favor to the dimeric MalDHs with more efficient and stringent function than LDH-like MalDHs was therefore a very likely means to prevent the selection of new LDHs from MalDH mutations.
Previously, the evolution of LDH from cytosolic MalDH (rooted within the dimeric group) was reported for T. vaginalis (Wu et al. 1999). This functional conversion has lead to an LDH that has conserved a high MalDH activity. In this study, we have observed that apicomplexan LDH enzymes were likely converted from a duplicated LDH-like MalDH gene originally acquired from an -proteobacterium (see node labeled "3" in figure 3B). This gene duplication led to the creation of a new type of LDH and was independent of the ancestral separation between LDH and LDH-like MalDHs (see node labeled "2" in figure 3A). In fact, these apicomplexan LDHs might be considered as LDH-like MalDH–resembling enzymes. Independently from this functional conversion of apicomplexan LDHs from an -proteobacterial MalDH, CpLDH1 and CpMalDH1 apparently evolved from the same ancestor (likely an MalDH1 gene) by a very recent event of gene duplication (see node labeled "4" in figure 3B).
Similar cases of functional conversion within the clade of dimeric MalDHs were described in kinetoplastids (Cazzulo Franke et al. 1999; Uttaro et al. 2000). In this clade, MalDH from Trypanosoma cruzi and Phytomonas have independently evolved twice to give new enzymes with a broader range of substrate specificity. In the case of Phytomonas the recent gene duplication of the glycosomal MalDH has lead to a 2-hydroxyacid dehydrogenase able to recognize phenyl pyruvate, 2-oxocaproate, and 2-oxoisocaproate in addition to oxaloacetate (Uttaro and Opperdoes 1997). All the functionnal conversions mentioned here suggest that, within the MalDHs (LDH-like and dimeric), the substrate recognition mechanism between dicarboxylic and monocarboxylic acid was bypassed at least four times during evolution. These events reflect the powerful organism plasticity in response to changes in their environments.
The different origin of CpLDH1 from other apicomplexan LDHs also provides additional evidence on the molecular and evolutionary divergence of C. parvum from other apicomplexans. Indeed, although conventional taxonomy has placed the Cryptosporidium genus within the Class Coccidea. mainly based on strong morphological evidence, several early phylogenetic reconstructions have clearly placed this genus at the base of the Apicomplexa (i.e., monophyletic to Coccidia + Haematozoa) (Morrison and Ellis 1997; Zhu, Keithly, and Philippe 2000) or as a sister to the gregarines (Carreno, Martin, and Barta 1999). Unlike other apicomplexans, C. parvum apparently lacks plastid and mitochondrial genomes, based on experimental data (Zhu, Marchewka, and Keithly 2000) or data mining the nearly complete genome project. All these observations suggest that Cryptosporidium has a unique evolutionary history that differs from other apicomplexans at both organismal and metabolic levels.
Acknowledgements
This research was supported in part by grants from the National Institute of Allergy and Infectious Diseases (NIAID) at the National Institutes of Health (NIH), U.S. Department of Human Health Services (DHHS) (R01 AI44594 and R21 AI055278 to G.Z. and U01 AI046397 to M.S.A.) and by a grant from the European Economic Community (EEC): "European network for biological deuteration HPRI CT 2001 50035" to D.M. Preliminary sequence data for Toxoplasma gondii malate dehydrogenase (MalDH) was obtained from The Institute for Genomic Research Web site at http://www.tigr.org. Sequencing of T. gondii genome was funded by NIAID. Preliminary sequence data for Eimeria tenella MalDH were produced by the E. tenella genome project at the Sanger Institute and can be obtained from ftp://ftp.sanger.ac.uk/pub/databases/E.tenella/, which was funded by the U.K. Biotechnology and Biological Science Research Council (BBSRC).
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