The mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase of Trypanosomatidae and the glycosomal redox balance of insect stages of Trypanosoma brucei and Leishmania spp.
Introduction
In organisms of the Trypanosomatidae family, the core carbohydrate metabolism, including a major part of the pathway for glycolysis and part of glycerol metabolism, takes place inside a specialized peroxisome, called the glycosome [1]. The membranes of these organelles are impermeable for many solutes including NAD(H) [2], [3], [4]. Therefore, this compartmentalization requires special mechanisms to route and regulate the metabolism of glucose and glycerol. In order to use glycerol as a carbon and energy source, the organisms need to first phosphorylate it and then oxidise the produced glycerol 3-phosphate (G3P) to dihydroxyacetone phosphate (DHAP) [5]. For this second step, it has been suggested that Leishmania mexicana uses the glycosomal NADH-dependent glycerol-3-phosphate dehydrogenase (GPDH, EC 1.1.1.8), thus allowing the usage of glycerol and/or G3P from triglycerides [6]. However, the equilibrium constant of the NAD-linked reaction favours the direction by which DHAP is reduced to G3P, as has been shown for the reaction catalyzed by the Leishmania enzyme in vitro [6] and observed in plants, yeasts and mammals [7], [8], [9]. Moreover, in the Trypanosoma brucei bloodstream form, this enzyme is known to work in the direction of G3P formation at a high rate, as part of a G3P shuttle that controls the NAD+/NADH ratio in the glycosome, by transferring the reductive equivalents from NADH, formed by glycolysis, to the mitochondrion and thus allowing a continued high glycolytic flux. This is similar to the mitochondrial G3P/DHAP shuttle observed in several organisms where it functions to regenerate cytosolic NAD+ and feed the respiratory chain for the production of ATP. In bloodstream-form T. brucei the reducing equivalents are transported as G3P from the glycosome to the mitochondrion where a glycerol 3-phosphate oxidase (GPO) retransforms it to DHAP [1]. This GPO is in fact a system comprising an FAD-dependent GPDH (EC 1.1.99.5), ubiquinone and an alternative oxidase (called TAO for trypanosome alternative oxidase). The equilibrium constant of the FAD-dependent reaction is such that the enzyme is able to oxidize G3P to DHAP, donating the electrons to the ubiquinone pool, which is in turn reoxidised with the concomitant reduction of oxygen to H2O by the TAO. The TAO represents the only terminal oxidase available in the bloodstream form since the respiratory complexes III and IV are absent in this life-cycle stage of T. brucei [10]. In this way, for every half-glucose equivalent (glyceraldehyde 3-phosphate) that is converted to 1,3-bisphosphoglycerate by the glyceraldehyde-3-phosphate dehydrogenase (GAPDH; enzyme 10 in Fig. 1), the other equivalent (DHAP) needs to pass through one shuttle cycle (i.e. glycosomal NADH-GPDH and mitochondrial FAD-GPDH; enzymes 7 and 9 in Fig. 1) in order to regenerate the consumed NAD+ and route the produced DHAP back to the glycosome where it is converted to glyceraldehyde 3-phosphate and further metabolised in the glycolytic pathway. The sole alternative to running a full shuttle cycle is to convert G3P into glycerol via the reverse reaction of the glycerol kinase (enzyme 8 in Fig. 1), but taking this path means to sacrifice the synthesis of one out of two molecules of ATP per molecule of glucose in the cytosol and trypanosomes are not able to sustain normal growth then [11], [12].
The case is different for the insect (procyclic) stage of T. brucei and other members of this family, where the presence of malate dehydrogenase [13], [14] and an NADH-dependent fumarate reductase in the glycosome [15] provides additional means for the re-oxidation of the NADH produced by glycolysis. At first sight, if we assume that 50% of the produced phosphoenolpyruvate (PEP) returns to the glycosome to be reduced in two NADH-dependent steps to succinate (see Fig. 1), this would leave the G3P shuttle as a superfluous pathway. However, a TAO activity is present in procyclics [16] and addition of G3P to isolated mitochondria led to a significant respiration rate [16], [17], [18]. These data, as well as previous experiments showing that G3P can be produced inside glycosomes without leading to significant glycerol production [19], are indicative of a functional shuttle in this stage too, but its possible contribution to the overall metabolism has not received much attention so far. Therefore, we embarked on a study to explore in more detail the existence of a functional shuttle and its role in carbohydrate metabolism of procyclic T. brucei. Studies on the procyclic stage are also relevant for Trypanosoma cruzi and Leishmania spp., because it is considered as a good model due to the metabolic similarity with these other related pathogens. Along the same line we also studied the L. major genes and the consequences of the inclusion of an active FAD-GPDH-coupled G3P/DHAP shuttle in the currently established metabolic schemes of the insect stages of both organisms.
Section snippets
Sequence analysis
The GUT2 gene of Saccharomyces cerevisiae encodes a mitochondrial FAD-dependent GPDH [20]. The Gut2p amino acid sequence (Swiss-Prot accession number P32191) was used to perform a BLAST search against sequences of all members of the Trypanosomatidae family available in the GeneDB databases (http://www.genedb.org). All sequences recognized were retrieved and stored locally. Multiple alignments were made with the Clustal X program, version 1.8, using BLOSUM matrix and default settings [21].
The
Sequence analysis
The amino acid sequence of S. cerevisiae Gut2p, the mitochondrial FAD-dependent GPDH, was used in a TBLASTN search against the GeneDB genomic databases in order to identify homologues of this protein in Kinetoplastida. A multiple alignment of putative FAD-GPDH sequences of Trypanosomatidae and other selected species is shown in Fig. 2. In Fig. 3 is shown that the T. brucei gene is expressed in both procyclic and bloodstream stage cells, as determined by RT-PCR.
A region corresponding to the
Discussion
We have proved the identity of a putative FAD-GPDH sequence in L. major by showing that the protein has the expected activity and that it is efficiently targeted to the mitochondrion. Most likely, this conclusion holds true also for the homologous sequences identified in the other Trypanosomatidae, because of their high similarity with the Leishmania one and much more certain predictions for mitochondrion-targeting signals. It should be noted that the sequences of mitochondrion-targeting
Acknowledgements
This research was supported through grants from the Interuniversity Attraction Poles by the Belgian Federal Office for Scientific, Technical and Cultural Affairs and from the European Commission through its INCO-DEV programme (contract ICA4-CT-2001-10075). DGG acknowledges a PhD scholarship from the ‘Commission de Coopération Universitaire au Développement, commission permanente du Conseil Interuniversitaire de la Communauté Française’ and an UNESCO-American Society of Microbiology Travel Award
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Present address: Department of Microbiology, Universidad Peruana Cayetano Heredia Av. Honorio Delgado 430, Lima 31, A.P. 4314, Perú.