Unique phylogenetic relationships of glucokinase and glucosephosphate isomerase of the amitochondriate eukaryotes Giardia intestinalis, Spironucleus barkhanus and Trichomonas vaginalis

Abstract

Glucokinase (GK) and glucosephosphate isomerase (GPI), the first two enzymes of the glycolytic pathway of the diplomonads Giardia intestinalis and Spironucleus barkhanus, Type I amitochondriate eukaryotes, were sequenced. GPI of the parabasalid Trichomonas vaginalis was also sequenced. The diplomonad GKs belong to a family of specific GKs present in cyanobacteria, in some proteobacteria and also in T. vaginalis, a Type II amitochondriate protist. These enzymes are not part of the hexokinase family, which is broadly distributed among eukaryotes, including the Type I amitochondriate parasite Entamoeba histolytica. G. intestinalis GK expressed in Escherichia coli was specific for glucose and glucosamine, as are its eubacterial homologs. The sequence of diplomonad and trichomonad GPIs formed a monophyletic group more closely related to cyanobacterial and chloroplast sequences than to cytosolic GPIs of other eukaryotes and prokaryotes. The findings show that certain enzymes of the energy metabolism of these amitochondriate protists originated from sources different than those of other eukaryotes. The observation that the two diplomonads and T. vaginalis share the same unusual GK and GPI is consistent with gene trees that suggest a close relationship between diplomonads and parabasalids. The intriguing relationships of these enzymes to cyanobacterial (and chloroplast) enzymes might reflect horizontal gene transfer between the common ancestor of the diplomonad and parabasalid lineages and the ancestor of cyanobacteria.

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.