*Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology, Department of Biochemistry, Université catholique de Louvain, Brussels, Belgium;
Institute of Evolutionary Biology, Department of Biology, Universität Konstanz, Konstanz, Germany;
Institut für Botanik, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany;
§The Rockefeller University, New York; and
||Institute of Marine Biosciences, National Research Council, Halifax, Nova Scotia, Canada
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Abstract |
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Introduction |
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In the vast majority of eukaryotes studied to date, glycolysis is a cytosolic pathway. This is always true in eukaryotes that lack organellar compartments involved in ATP synthesis, such as Giardia lamblia (Müller 1998
) or Mastigamoeba balamuthi, an amoeboflagellate protist that possesses a strikingly barren cytoplasm and lacks morphologically recognizable mitochondria (Chavéz, Balamuth, and Gong 1986
; Brugerolle 1993
). However, among other protists, there are some notable exceptions to be found with regard to the compartmentation of glycolysis. In Chlamydomonas reinhardtii, for example, all glycolytic enzyme activites studied to date were found to be localized in the chloroplast, rather than in the cytosol (Schnarrenberger et al. 1990
). A better-known and well-studied exception is the kinetoplastids (trypanosomes and related organisms), in which most of the glycolytic pathway is compartmentalized in specialized microbodies, glycosomes (Opperdoes and Borst 1977
; Hannaert and Michels 1994
; Clayton and Michels 1996
). In these organisms, only the last three enzymes of the pathway leading to pyruvate production, including enolase, are found in the cytosol. In relatives of the kinetoplastids, the euglenids, which possess plastids but not glycosomes, some of the enzymes that are common to glycolysis and the Calvin cycle occur as distinct chloroplast-cytosol isoenzyme pairs (Kitaoka et al. 1989
; Henze et al. 1995
; Plaumann et al. 1997
), as is also found in many higher plants (Martin and Schnarrenberger 1997
). Several key glycolytic enzymes from trypanosomes and amitochondriate protists differ in their regulatory properties from those of other eukaryotes, e.g., yeast or vertebrates, differences that appear to correlate with the lifestyle of these organisms (Mertens, van Schaftingen, and Müller 1992
; Mertens 1993
; Hannaert and Michels 1994
; Michels and Hannaert 1994; Clayton and Michels 1996
; Bakker et al. 1997
; Park, Schofield, and Edwards 1997
).
The study of the evolution of pathways requires sequences for all of the enzymes involved. For the kinetoplastid Trypanosoma brucei, all glycolytic enzymes except enolase have been cloned. For amitochondriate protists and Euglena, whose glycolytic pathways differ from kinetoplastids not only with respect to compartmentation, but also with respect to regulation of the enzymes involved (Kitaoka et al. 1989
; Bakker et al. 1997
), fewer sequences for glycolytic enzymes are known. Here we report the molecular analysis of cytosolic enolase from the amitochondriate protist M. balamuthi and T. brucei and two isoenzymes of enolase from Euglena gracilis, one of which possesses a putative N-terminal plastid-targeting peptide. A protein phylogeny of enolase sequences from eubacterial, archaebacterial, and eukaryotic sources is presented that reveals insights into the complex evolutionary history and subcellular compartmentation of this glycolytic enzyme.
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Material and Methods |
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Cloning and Molecular Analysis of E. gracilis Enolase Isoenzymes
Axenic E. gracilis cultures (SAG 1224-5/25) were grown as described (Henze et al. 1995
) under a 14:10 h light : dark regime. Isolation of mRNA through two rounds of oligo-dT cellulose chromatography and synthesis of cDNA for amplification was performed as described (Henze et al. 1995
) but without the NotI adapters. Degenerate primers 1F (5'-GAYWSIMGIGGIAAYCCIACIGTIGAR-3') and 3R (5'RTCYTCIGTYTCICCISWICKRTG-3') were designed against the conserved amino acid sequence motifs "DSRGNPTVE" and "HRSGETED" from the N-terminal and C-terminal regions, respectively, of an alignment of enolase sequences extracted from GenBank. Amplification was performed for 35 cycles of 2 min at 94°C, 1 min at 50°C, and 2 min at 72°C in 25 µl containing 10 µM of each primer, 50 µM dNTP, 1 mM MgCl2, 10 ng cDNA, and 0.5 U Taq polymerase (Perkin-Elmer) using the supplier's buffer. The 1.1-kb fragment was subcloned and used to screen 300,000 recombinants of a Euglena cDNA library (Henze et al. 1995
). From 150 hybridizing positives, 15 were subcloned and sequenced. One corresponded to pEgEno29. The other 14 represented an mRNA encoding a different enolase sequence, the largest clone of which was designated pEgEno02.
For Northern blots, inserts of pEgEno02 and pEgEno29 were purified by electroelution. Five micrograms of polyA+ mRNA purified through two rounds of oligo-dT cellulose affinity chromatography was loaded per lane on the same formaldehyde gel, electrophoresed as described (Sambrook, Fritsch, and Maniatis 1989
), and transferred to Hybond N (Amersham) membranes according to the manufacturer's protocol. Lanes were stained, photographed, cut, and hybridized separately at 70 °C in 3 x SSPE, 0.1% SDS, 0.02% (w/v) PVP, 0.02% (w/v) Ficoll 400, 50 µg/ml polyadenylic acid, and 10 ng/ml of probe labeled to 5 x 107 cpm/µg. Washing was at 70 °C in 2 x SSPE, 0.1% SDS.
Spinach Enolase
Using cDNA from 10-day-old spinach seedlings, the primers 1F and 3R, described above for Euglena, were used under the same conditions to amplify a 1.1-kb fragment from spinach. The amplification product was cloned as above and used to screen a spinach cDNA library prepared as described (Nowitzki et al. 1998
). Ten independent positive clones were identified, subcloned, and shown by terminal sequencing to represent the same mRNA. The longest of these, pSoEno8.3, was sequenced.
Enolase from M. balamuthi
Mastigamoeba balamuthi identical to the American Type Culture Collection isolate ATCC 30984, was obtained from Dr. Frederick Schuster (Brooklyn College of the City University of New York, Brooklyn, N.Y.). cDNA clone PHR60, containing the complete ORF for enolase, was identified in our ongoing M. balamuthi cDNA sequencing project, to be described elsewhere. The insert in this clone was sequenced on both strands by primer walking.
Sequence Handling and Phylogenetic Analysis
From sequences extracted from GenBank, an alignment was produced with CLUSTAL W (Thompson, Higgins, and Gibson 1994
) that was manually refined using the program ED of the MUST package (Philippe 1993
). Regions of uncertain alignment were omitted from the analysis, leaving 400 amino acid positions for analysis. Protein phylogeny was inferred using protein maximum-likelihood (ML) as implemented in MOLPHY, version 2.3 (Adachi and Hasegawa 1996
), using local rearrangement starting with the neighbor- joining (NJ) tree and using the best tree obtained from 1,000 PROTML topologies generated using the quicksearch option of MOLPHY using the JTT-F model. Distance analyses were performed with the MUST package using the Kimura correction and the NJ method (Saitou and Nei 1987
) using 1,000 bootstrap replicates. Parsimony analysis was performed using PAUP* with 500 bootstrap replicates and 10 times random addition (Swofford 1999
). The significance of different ML topologies was tested using the Kishino-Hasegawa test implemented in the MOLPHY package. Accession numbers for all sequences and the alignment are available via anonymous ftp from 134.169.70.80/ftp/pub/incoming/enolase.
Molecular Modeling
The structure of yeast enolase (Wedekind, Reed, and Rayment 1994
) was downloaded from the Protein Data Bank (http://www.rcsb.org/pdb/) and visualized using RASMOL (http://www.umass.edu/microbio/rasmol/) on a Macintosh computer.
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Results |
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Enolase from the Amitochondriate Protist M. balamuthi
Of the approximately 200 clones sequenced from a random cDNA library of M. balamuthi, six clones encoded a typical enolase. Of these, one clone was completely sequenced and found to code for a putative enolase of 438 amino acid residues with a molecular mass of 47,596, a pI of 6.7, and a net charge of -3.
Cytosolic and Plastid-Targeting Peptide-Bearing Isoforms of Enolase in Euglena
Grown in rich medium in the light under aerobic conditions, Euglena expresses two different genes for enolase. The sequence of pEgEno02 is 1,523 bp long, including a 21-base polyA stretch, and encodes an ORF of 431 amino acids with a molecular mass of 46,501, a pI of 5.79, and a net charge of -7. pEgEno29 is 1,622 bp long with a 10-base polyA tail and encodes an ORF of 485 amino acids that lacks a start codon near the N-terminus of pEgEno02. Instead, the reading frame extends beyond the start codon of pEgEno02 by at least 53 amino acids. This region extends into, but terminates in, a highly hydrophobic region (fig. 1
). The plastid targeting-peptides of nuclear-encoded chloroplast proteins in Euglena are roughly 120150 amino acids long and possess two highly hydrophobic regions (Plaumann et al. 1997
) for transport across the three membranes that surround the plastids of Euglena (Schwartzbach, Osafune, and Löffelhardt 1998
). The position of the proximal hydrophobic region in the protein encoded by pEgEno29 relative to the N-terminus of other enolases is very similar to the position noted in the plastid-targeting peptide of Euglena chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Henze et al. 1995
), suggesting that this cDNA might encode a chloroplast isoform. Assuming the processing site indicated in figure 1
, the molecular mass of the mature protein would be 46,751, and its pI would be 7.12, with a net charge of -1.
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Discussion |
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The first of these, at positions 161165 of our alignment, underscores the specificity of a fiveamino acid insertion previously noted to be shared in cytosolic enolase from higher plants and enolase from the apicomplexans Plasmodium falciparum (Read et al. 1994
) and Toxoplasma gondii (Dzierzinski et al. 1999
). This indel suggests that apicomplexan parasites, which possess plastids (McFadden et al. 1996
; McFadden and Roos 1999
), probably obtained their nuclear enolase gene from the photosynthetic symbiont (Read et al. 1994
; Dzierzinski et al. 1999
), a view that receives additional support from the current analysis, due to the highly restricted phylogenetic distribution of the indel.
Interestingly, two enolase sequences from plants, that from Chlamydomonas and a new enolase sequence from the Arabidopsis genome project (Arabidopsis 2 in fig. 3
), do not share the insertion at positions 161165. The Arabidopsis 2 protein also carries a transit peptide, suggesting that it represents a plastid isoform. Many higher plant tissues are known to possess distinct and separable chloroplast-cytosol isoenzymes of enolase, for example, developing Ricinus seeds (Miernyk and Dennis 1992
) and developing barley leaves (Hoppe et al. 1993
), but the plastid enzyme is usually not highly expressed in fully green tissues. A previous study indicated the lack of a plastid enolase in Arabidopsis (van der Straeten et al. 1991
), but it seems likely that the plastid enzyme is expressed only in early stages of Arabidopsis development, as is the case in barley (Hoppe et al. 1993
). Like the probable plastid isoform from Arabidopsis, the Chlamydomonas enolase also lacks the fiveamino acid insertion characteristic of plant (and apicomplexan) cytosolic enolase. Furthermore, a study of subcellular distribution of several glycolytic enzymes in Chlamydomonas has shown that only a single isoform for each of the glycolytic enzymes glucose-6-phosphate isomerase (GPI), FBA, phosphoglycerate kinase (PGK), and TPI exists and that each is localized in the chloroplast (Schnarrenberger et al. 1990
). Although enolase was not assayed in that study, the lack of cytosolic isoenzymes for those glycolytic activities indicates that glycolysis in light-grown Chlamydomonas is primarily localized in the plastid, suggesting that the Chlamydomonas enolase is also a plastid isoform, consistent with its shared lack of the insertion at positions 161165. Since the Chlamydomonas enolase sequence in the database lacks about 50 residues of the mature subunit, it is possible that the complete clone may possess a transit peptide.
Another notable indel occurs at positions 305306 of the alignment. All enolase sequences from eukaryotic sources lack the motif "PG" (or its variants) at this position, with the notable exception of the Euglena isoenzymes. All prokaryotic sequences surveyed possess the PG motif, with the exception of the archaebacterium Methanococcus and the eubacterium Campylobacter. However, the positions of Methanococcus, Campylobacter, and eukaryotic enolases in the gene phylogeny (fig. 4 ) suggest that this indel does not represent a unique event in enolase evolution, but rather that this deletion has occurred several times independently. That independent indel events can occur at this position is furthermore seen by the unique event in the Chlorobium 1 sequence (fig. 3 ).
Conflict Between Indel Distribution and Protein Phylogeny in Plasmodium Enolase
The conspicuous indel in figure 3
that indicates common ancestry of the Plasmodium and plant cytosolic enolases is not consistent with the position of Plasmodium in the phylogeny of figure 4
. Instead of branching with plant cytosolic enolase, the Plasmodium sequence tends to assume a basal position among eukaryotic homologs. Also in distance and parsimony analyses, the Plasmodium sequence did not branch with the plant homologs, but rather assumed a basal position, whereby it branched above the branch bearing Trypanosoma, Mastigamoeba, and Entamoeba in parsimony analysis and branched with Trypanosoma and Entamoeba when Mastigamoeba was excluded in PROTML.
The nature of the indel at positions 161165 (fig. 3
) suggests that Plasmodium enolase does indeed share a common ancestry with the plant cytosolic sequences and that its position in figure 4
may be a reconstruction artifact. Overall, the position of the Plasmodium sequence was rather unstable, and it seems likely that its basal position in figure 4
is due to a long-branch attraction artifact and is hence a misplacement. Using the Kishino-Hasegawa test, PROTML trees in which Plasmodium was forced with the plant sequences were not worse than the tree in figure 4
at the 5% level. In parsimony analyses of smaller data sets, consisting of 45 sequences through exclusion of many eubacterial sequences, the Plasmodium sequence shared a common branch with its homologs from higher plants, but with a bootstrap proportion of only 46%. In an analysis of the enolase sequence from the apicomplexan T. gondii, Dzierszinski et al. (1999)
also found that with smaller data sets, the apicomplexan enolase sequences branched with plant homologs in parsimony analyses.
Among the 585 trees found with the quicksearch option of MOLPHY that were not significantly worse at the 5% level than the best tree found, only 23 grouped Plasmodium with the plant sequences. The position of Plasmodium enolase is thus difficult to resolve in phylogenetic inference from sequence data, with the conflict between the specific indel (fig. 3
) and the position of Plasmodium enolase in the phylogeny suggesting that it is simply misplaced in figure 4
. In light of the plastid that has been found in apicomplexans (McFadden et al. 1996
; McFadden and Roos 1999
), a link between apicomplexan and plant enolase, as evidenced by the indel at positions 161165, is not surprising. Indeed, in analyses of a different glycolytic enzyme, GPI, sequences from apicomplexans did branch specifically with their plant homologs in PROTML analyses (Nowitzki et al. 1998
; Dzierszinski et al. 1999
).
Enolases from Amitochondriate Protists Branch with the Trypanosoma Homolog
The detection of enolase transcripts indicates that the core metabolism of M. balamuthi involves an Embden-Meyerhof-Parnastype glycolysis. This conclusion is supported by the identification of cDNA clones from the EST project encoding PPi-linked phosphofructokinase (PPi-PFK), class II fructose1,6-bisphosphate aldolase, GAPDH, and phosphoglycerate mutase (PGM) (unpublished data). The organism contains no mitochondrion- or hydrogenosome-like structures (Chavéz, Balamuth, and Gong 1986
; Brugerolle 1993
) and grows in a cysteine-rich anaerobic medium, and lactate is one of its major end products (unpublished data). These characteristics permit an assignment of M. balamuthi to type I amitochondriate protists characterized by lack of compartmentalized energy metabolism (Martin and Müller 1998
; Müller 1998
).
Mastigamoeba balamuthi is a member of the amoeboflagellate group of pelobionts, which also contains the giant amitochondriate amoeba Pelomyxa palustris. Because of its simple cytological makeup, this group of eukaryotes is often regarded as "primitive" and is placed with other mitochondrion-lacking eukaryotes in some taxonomic schemes (Cavalier-Smith 1991, 1993
). The evolutionary position of these organisms, however, remains unresolved (Embley and Hirt 1998
). While some data indicate an ancestral (basal) position of this group among eukaryotes (Stiller, Duffield, and Hall 1998
), others suggest a derived, nonbasal position (Hinkle at al. 1994
). The Mastigamoeba sequence branches with Trypanosoma and Entamoeba enolase also in distance and parsimony analyses. The limited taxonomic sample provides no further insight into the evolution of enolase in these protists.
Gene Duplications for the Origin of Chloroplast-Cytosol Enolase Isoenzymes
As discussed above, available molecular and biochemical data suggest that the putative transit peptide-bearing enolases from Arabidopsis and Chlamydomonas likely represent plastid isoforms of the enzyme. The position of these isoforms in figure 4
suggests that the genes for the chloroplast-specific isoforms were not acquired by the nucleus from the cyanobacterial ancestor of plastids, but, rather, arose through gene duplication involving the cytosolic isoform and acquisition of a transit peptide, as is the case for many compartment-specific enzymes that were common to hosts and symbionts during the origin of organelles (Martin and Schnarrenberger 1997
). This duplication appears to have taken place relatively early in plant evolution, probably prior to the origin of the insertion at positions 161165 that is specific to the plant cytosolic isoforms (under the reasonable premise that the Plasmodium sequence is misplaced in the phylogenetic analyses).
Enolase from Euglena Is an Exception
Many genes studied to date underscore a common ancestry of trypanosomes and euglenids. These include nuclear-encoded ribosomal RNA (Sogin 1994
), tubulin (Levasseur, Meng, and Bouck 1994
), Hsp60 (Yasuhira and Simpson 1997
), and the ER-specific protein calreticulin (Navazio et al. 1998
), in addition to mitochondrial-encoded cytochrome oxidase subunit I (Tessier et al. 1997
; Yasuhira and Simpson 1997
). Enolase is thus an exception in that it clearly does not reflect the common ancestry of the kinetoplastid (Trypanosoma) and euglenid lineages (fig. 4
).
In contrast to trypanosomes, euglenids possess plastids. These are surrounded by three membranes, suggesting that Euglena may have acquired its plastids by secondary symbiosis through engulfment of a eukaryotic green alga (Gibbs 1978
), a view that is supported by chloroplast genome phylogeny (Martin et al. 1998
). As in the case of higher plants and Chlamydomonas, the cytosolic and plastid-targeting peptide-bearing forms of Euglena enolase also must have arisen through gene duplication. Yet, the source of the eukaryotic nuclear gene which gave rise to that duplication is different from that of all other eukaryotic enolase genes. Enolase genes in Euglena are much more similar to the homolog from the spirochaete Treponema than they are to enolase from any other source currently sampled. This is a mildly surprising, but by no means unprecedented, finding. The nuclear gene for cytosolic GAPDH in Euglena also branches quite robustly with its homolog from the Treponema genome (Figge et al. 1999
). Further affinities between spirochaete and euglenozoan (kinetoplastid and euglenid) glycolytic enzymes are found for PFK, for which the Trypanosoma homolog branches with Borrelia and, curiously, as with enolase, Entamoeba homologs (Michels et al. 1997
; Mertens et al. 1998
).
At face value, the surprising affinity of nuclear-enoded Euglena enolases to the Treponema homolog could reflect an outright lateral (i.e., nonendosymbiotic) gene transfer from spirochaetes to the Euglena lineage. If so, then future sampling of eukaryotic groups should reveal that the acquisition is specific to the euglenid lineage. If, on the other hand, other, more distantly related, eukaryotic lineages are also found to possess enolase genes that branch with the Treponema homolog, then the acquisition could be traced more deeply into eukaryotic phylogeny, in which case differential loss could be invoked to explain the distribution of enolase genes among eukaryotes. In analogy, a similarly "odd" aldolase gene was recently found in Giardia that is closely related to -proteobacterial homologs, whereas other eukaryotic aldolase genes described to date are not (Henze et al. 1998
). As in the case of Euglena enolase, it is possible that other eukaryotes will be found to possess the same "odd" FBA gene as Giardia does.
Thus, further sampling of protist lineages is needed before a strong case for or against horizontal enolase gene transfer from prokaryotes to Euglena in a context that did not involve the origin of organelles can be argued. Several previous claims of outright prokaryote-eukaryote horizontal gene transfer (i.e., not involving the origins of chloroplasts or mitochondria) argued on the basis of small species samples have turned out, with time, to be just as easily or more easily explained by gene transfer in the context of the origins of organelles (Martin and Schnarrenberger 1997
; Nowitzki et al. 1998
), warranting caution when only few protist sequences are available for comparison. The finding that prokaryotes tend to exchange genes via horizontal transfer (Lawrence and Ochman 1998
; Vorholt et al. 1999
) lends an additional degree of complexity to the problem of trying to identify the source of eukaryotic nuclear genes (Brown and Doolittle 1997
; Martin and Schnarrenberger 1997
; Doolittle 1999
). The non-rRNA-like phylogeny of some eubacterial enolases in figure 4
for example, a lack of common branching for the two Chlorobium enolases, the sequences from two spirochaetes, or the four sequences from low-GC Gram positiveswould not exclude the possibility that some transfer of enolase genes among eubacteria has occurred.
Eukaryotic Enolase Is an Exception Among Glycolytic Enzymes
Previous phylogenetic studies of glycolytic/gluconeogenetic enzymes have revealed that most of the glycolytic pathway in the eukaryotic cytosol consists of enzymes that are more similar to eubacterial homologs than they are to archaebacterial homologs. This is true for GAPDH (Markos, Miretsky, and Müller 1993
; Henze et al. 1995
; Wiemer et al. 1995
), PGK (Brinkmann and Martin 1996
; Adjé, Opperdoes, and Michels 1998
), FBA (Plaumann et al. 1997
; Henze et al. 1998
), FBP (Martin and Schnarrenberger 1997
), TPI (Keeling and Doolittle 1997
), PFK (Siebers, Klenk, and Hensel 1998
; Mertens et al. 1998
), and GPI (Nowitzki et al. 1998
).
In marked contrast to those enzymes, eukaryotic enolase sequences are not more similar to eubacterial than to archaebacterial homologs, with the exception of Euglena. Rather, they tend to branch near the homologs from Pyrococcus horihoshii, Pyrococcus furiosus, and Methanococcus jannaschii. However, the position of the branch bearing these three archaebacterial sequences basal to eukaryotic enolase in figure 4 is not strongly supported, and the deeper branches of the enolase protein phylogeny are quite difficult to resolve. For example, among the 585 trees found using the quicksearch option of MOLPHY that were not significantly worse than the best tree at the 5% level, 52 placed the six archaebacterial sequences on a single common branch and 82 placed the branch bearing the Euglena and Treponema sequences basal to the eukaryotic homologs.
In this respect, it is notable that Methanococcus enolase, like the eukaryotic sequences, lacks the insertion at positions 305306 in figure 3
, which at face value might tend to favor an archaebacterial ancestry of the eukaryotic enzyme. However, this polymorphism is not phylogenetically unique among prokaryotes, since it is also found in the Campylobacter sequence (fig. 3
), suggesting that it may have occurred in parallel in independent lineages. To examine this possibility in further detail, we mapped the corresponding position to the three-dimensional structure of enolase determined from yeast (Wedekind, Reed, and Rayment 1994
). The residues of the yeast enzyme that correspond to the position of the indel in question are Asp238, Gly239, and Lys240 in the numbering of Wedekind, Reed, and Rayment (1994)
, which are numbered 304, 317, and 318 in figure 3
. As shown in figure 5 , these positions map to a small loop at the surface of the yeast enolase dimer, such that insertions and deletions at this position might be easily tolerated by the proteins without altering enzyme function. The additional 10 residues that are found at this position in the Chlorobium 1 sequence (fig. 3
) may thus form a small external loop. That the plant- and apicomplexan-specific insertion at positions 161165 (fig. 3
) also maps to the surface of the enzyme, where it likely forms a small external loop, was recently shown by comparative modeling with the yeast enolase structure (Dzierszinski et al. 1999
).
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Conclusions |
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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1 Abbreviations: FBA, fructose-1,6-bisphosphate aldolase; FBP, fructose-1,6-bisphosphatase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPI, glucose-6-phosphate isomerase; ORF, open reading frame; PGK, phosphoglycerate kinase; PCR, polymerase chain reaction; PFK, phosphofructokinase; PGM, phosphoglycerate mutase; PPi-PFK, PPi-linked phosphofructokinase; PVP, polyvinylpyrrolidone; SDS, sodium dodecyl sulphate; TPI, triosephosphate isomerase.
2 Keywords: glycolysis
metabolism
amitochondriate protist
phylogeny
evolution
organelles
endosymbiosis
3 Address for correspondence and reprints: William Martin, Institut für Botanik, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany. E-mail: w.martin{at}tu-bs.de
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