Unique phylogenetic relationships of glucokinase and glucosephosphate isomerase of the amitochondriate eukaryotes Giardia intestinalis, Spironucleus barkhanus and Trichomonas vaginalis
Introduction
Catabolic core metabolism refers to the conversions of low molecular weight organic molecules either derived from nutrients taken up by the cell or stored inside the cell providing free energy and intermediates for diverse life processes. A key example of such processes is glycolysis, the conversion of a hexose to three- or two-carbon endproducts with the production of a limited amount of ATP per hexose converted (Fothergill-Gilmore and Michels, 1993). This pathway is regarded as rather stereotyped in eukaryotes but a number of variations on this theme have been detected in diverse prokaryotes. In fact, a number of prokaryotes are known to lack this process completely or partially (Dandekar et al., 1999).
While glycolysis in eukaryotes is uniformly represented by the Embden–Meyerhof–Parnas (EMP) type of the pathway, certain formally identical steps can be catalyzed by enzymes that differ from each other in diverse eukaryotes (Fothergill-Gilmore and Michels, 1993, Michels et al., 2000, Müller, 1998, Wu et al., 2001). Although this diversity is by far smaller than the diversity seen among prokaryotes (Dandekar et al., 1999), it still is considerable and suggests a complex evolutionary history of the process in diverse eukaryotic lineages. This makes a systematic study of glycolytic enzymes in various eukaryotes a promising enterprise.
Studies on eukaryotes that represent biochemical variations on the basic theme of EMP glycolysis permit the exploration of the boundaries of possible diversity (Müller, 1998). The most extreme variation in biochemical makeup is found in the ‘amitochondriate’ eukaryotes, a group of unrelated organisms that differ from other eukaryotes by lacking a structure with the energy conserving functions of mitochondria (Martin and Müller, 1998, Müller, 1998) and relying on extended glycolysis as the core of their energy metabolism (Müller, 1998). These organisms belong to two major types according to the subcellular organization of their energy metabolism. Type I amitochondriates have no metabolic compartmentation while Type II organisms have a cytosolic/hydrogenosomal one (Martin and Müller, 1998, Müller, 1998). The metabolism and metabolic enzymes of only a few parasitic species of ‘amitochondriate’ protists (primarily the diplomonad Giardia intestinalis (syn. G. lamblia) and the entamoebid Entamoeba histolytica, two Type I organisms, and the parabasalid Trichomonas vaginalis, a Type II species), have been explored, but the data already available reveal a complex picture, far exceeding that observed in ‘mitochondriate’ eukaryotes. These organisms also exhibit major biochemical differences among each other.
The phylogenetic relationships of the glycolytic enzymes of these protists also show a complex picture, indicating past gene replacements, possibly due to lateral gene transfers. These conclusions are based, however, on the study of only a limited number of enzymes of a few species. To obtain a clearer picture and, more importantly, to discern the evolutionary events responsible for this complexity, more enzymes need to be explored in more species. The ongoing genome project on G. intestinalis (McArthur et al., 2000) and EST projects on a second diplomonad, the fish parasite Spironucleus barkhanus, and on T. vaginalis gave us the opportunity to add information on the first two enzymes of glycolysis, glucokinase (GK) and glucose-6-phosphate isomerase (GPI). Here we report the sequence of GK from the two diplomonads, thereby complementing earlier data for this enzyme from T. vaginalis (Mertens and Müller, 1990, Wu et al., 2001) and the sequences of GPI from the three species. We also describe the heterologous expression of G. intestinalis GK. The data indicate that these three ‘amitochondriate’ protists obtained their GK and GPI genes from a different, but possibly common, source than other eukaryotes. This source may have shared a most recent common ancestor with cyanobacteria and chloroplasts.
Section snippets
Organisms
Giardia intestinalis (syn. G. lamblia), strain WB, axenic clone 6 (corresponding to ATCC 30957), Spironucleus barkhanus, strain NOR-1A (ATCC 50380), and Trichomonas vaginalis axenic clone G3 (from Professor G.H. Coombs, University of Glasgow, UK) were used in this study.
Isolation of clones and sequencing of the G. intestinalis genes
Probes for the isolation of the genes for G. intestinalis GK and GPI were polymerase chain reaction (PCR) products obtained with the use of non-degenerate PCR primers. These were designed based on single run gDNA sequences with
Sequences
The G. intestinalis GK coding sequence in clone pGLgk9 was 1029 bp long with a G+C content of 55% and encoded a protein of 343 amino acids with a molecular mass of 38 kDa. The S. barkhanus GK EST contained an incomplete open reading frame (ORF) with a G+C content of 40%, corresponding to a 330 amino acid long open reading frame. Alignment with the G. intestinalis GK sequence indicated that six amino-terminal amino acid residues were potentially missing. The molecular mass of the putative
Acknowledgements
Authors thank Drs. A. McArthur, H.G. Morrison and M.L. Sogin (Josephine Bay Paul Center in Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, MA) for advice, discussion given and access to the search programs of the Giardia genome project (www.mbl.edu/giardia). The G. intestinalis genomic library was kindly provided by Drs. F.D. Gillin and S.B. Aley (University of California Medical School, San Diego, CA). Supported by National Institutes of Health grant AI
References (29)
- et al.
Glucokinase of Escherichia coli: Induction in response to the stress of overexpressing foreign proteins
Arch. Biochem. Biophys.
(1995) - et al.
A kingdom-level phylogeny of eukaryotes based on combined protein data
Science
(2000) - et al.
Convergent evolution of similar enzymatic function on different protein folds: The hexokinase, ribokinase, and galactokinase families of sugar kinases
Protein Sci.
(1993) - et al.
Evolution and regulatory role of the hexokinases
Biochim. Biophys. Acta
(1998) - et al.
Pathway alignment: application to comparative analysis of glycolytic enzymes
Biochem. J.
(1999) Glucose transport in Escherichia coli
FEMS Microbiol. Lett.
(1989)- et al.
Evolution of glycolysis
Prog. Biophys. Mol. Biol.
(1993) - et al.
Sequence and phylogenetic position of a class II aldolase gene in the amitochondriate protist, Giardia lamblia
Gene
(1998) - et al.
Chaperonin 60 phylogeny provides further evidence for secondary loss of mitochondria among putative early-branching eukaryotes
Mol. Biol. Evol.
(2001) - et al.
A single eubacterial origin of eukaryotic pyruvate:ferredoxin oxidoreductase genes: Implications for the evolution of anaerobic eukaryotes
Mol. Biol. Evol.
(1999)
MrBayes: Baysian Inference of Phylogeny
Energy metabolism of the anaerobic protozoon Giardia lamblia
Mol. Biochem. Parasitol.
The hydrogen hypothesis of the first eukaryote
Nature
The Giardia genome project database
FEMS Microbiol. Lett.
Cited by (54)
The UDP-glucose pyrophosphorylase from Giardia lamblia is redox regulated and exhibits promiscuity to use galactose-1-phosphate
2015, Biochimica et Biophysica Acta - General SubjectsCitation Excerpt :The possible metabolism of Gal in G. lamblia via the reaction catalyzed by GlaUDP-Glc PPase has as a major problem the absence of galactokinase in the parasite. Previous works reported that glucokinase (EC 2.7.1.2) [50] and phosphoglucomutase (PGM; EC 5.4.2.2) [51] present in this organism exhibit deeply distinctive functional properties. Also, it is worth to consider that Giardia is an early diverging eukaryote with many unusual features of ultrastructure, metabolism and gene sequence [7].
A unique hexokinase in cryptosporidium parvum, an apicomplexan pathogen lacking the krebs cycle and oxidative phosphorylation
2014, ProtistCitation Excerpt :Collectively, enzymes within the pathway such as hexokinases may serve as attractive drug targets in pathogens that rely solely or mainly on glycolytic pathway for producing ATP, but may be less suitable as drug targets in pathogens with a fully functional Krebs cycle and the capability of using alternative energy sources. Hexokinases have been explored as drug targets in Trypanosoma, Entamoeba and Trichomonas (Chambers et al. 2008; Henze et al. 2001; Hudock et al. 2006; Saavedra et al. 2007; Sanz-Rodriguez et al. 2007). A number of classes of inhibitors against Trypanosoma parasites have also been recently developed (Chambers et al. 2008; Hudock et al. 2006; Sanz-Rodriguez et al. 2007).
Representational difference analysis identifies specific genes in the interaction of Giardia duodenalis with the murine intestinal epithelial cell line, IEC-6
2012, International Journal for ParasitologyNaegleria gruberi metabolism
2011, International Journal for ParasitologyCitation Excerpt :This explains the presence of a separate fructokinase. The predicted N. gruberi glucokinase is related to the glucokinases of the trypanosomatids Trypanosoma cruzi and Leishmania major and of Trichomonas vaginalis and Giardia intestinalis, all closely related to the glucokinase of bacteria (Henze et al., 2001; Wu et al., 2001; Cáceres et al., 2007). Second, a classical ATP-dependent phosphofructokinase (PFK) is absent in N. gruberi and the second phosphorylation step of glycolysis is catalysed by a pyrophosphate- (PPi-)dependent PFK, with 82% identity to the sequence published for N. fowleri (Mertens et al., 1993).
- 1
Present address: Institute of Botany III, Heinrich-Heine-University of Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany.
- 2
Present address: Department of Medical Zoology, Kagawa Medical University, 1750-1 Ikenobe, Miki-cho, Kagawa 761-0793, Japan.