The mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase of Trypanosomatidae and the glycosomal redox balance of insect stages of Trypanosoma brucei and Leishmania spp.

https://doi.org/10.1016/j.molbiopara.2006.05.006Get rights and content

Abstract

The genes for the mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase were identified in Trypanosoma brucei and Leishmania major genomes. We have expressed the L. major gene in Saccharomyces cerevisiae and confirmed the subcellular localization and activity of the produced enzyme. Using cultured T. brucei procyclic and Leishmania mexicana promastigote cells with a permeabilized plasma membrane and containing intact glycosomes, it was shown that dihydroxyacetone phosphate is converted into pyruvate, and stimulates oxygen consumption, indicating that all components of the glycerol 3-phosphate/dihydoxyacetone phosphate shuttle between glycosomes and mitochondrion are present in these insect stages of both organisms. A computer model has been prepared for the energy and carbohydrate metabolism of these cells. It was used in an elementary mode analysis to get insight into the metabolic role of the shuttle in these insect-stage parasites. Our analysis suggests that the shuttle fulfils important roles for these organisms, albeit different from its well-known function in the T. brucei bloodstream form. It allows (1) a high yield of further metabolizable glycolytic products by decreasing the need to produce a secreted end product of glycosomal metabolism, succinate; (2) the consumption of glycerol and glycerol 3-phosphate derived from lipids; and (3) to keep the redox balance of the glycosome finely tuned due to a highly flexible and redundant system.

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

References (60)

  • L.J. Brown et al.

    Sequence of rat mitochondrial glycerol-3-phosphate dehydrogenase cDNA. Evidence for EF-hand calcium-binding domains

    J Biol Chem

    (1994)
  • D.A. Lehn et al.

    The sequence of a human mitochondrial glycerol-3-phosphate dehydrogenase-encoding cDNA

    Gene

    (1994)
  • J.J. Blum

    Intermediary metabolism of Leishmania

    Parasitol Today

    (1993)
  • S. Besteiro et al.

    Energy generation in insect stages of Trypanosoma brucei: metabolism in flux

    Trends Parasitol

    (2005)
  • J. Blattner et al.

    The 3′-untranslated regions from the Trypanosoma brucei phosphoglycerate kinase-encoding genes mediate developmental regulation

    Gene

    (1995)
  • G. Lenaz

    A critical appraisal of the mitochondrial coenzyme Q pool

    FEBS Lett

    (2001)
  • C. Nihei et al.

    Trypanosome alternative oxidase as a target of chemotherapy

    Biochim Biophys Acta

    (2002)
  • W.U. Ajayi et al.

    Site-directed mutagenesis reveals the essentiality of the conserved residues in the putative di-iron active site of the trypanosome alternative oxidase

    J Biol Chem

    (2002)
  • V. Coustou et al.

    ATP generation in the Trypanosoma brucei procyclic form: cytosolic substrate level is essential, but not oxidative phosphorylation

    J Biol Chem

    (2003)
  • J. Fang et al.

    Alternative oxidase present in procyclic Trypanosoma brucei may act to lower the mitochondrial production of superoxide

    Arch Biochem Biophys

    (2003)
  • T.N. Darling et al.

    A comparative study of d-lactate, l-lactate and glycerol formation by four species of Leishmania and by Trypanosoma lewisi and Trypanosoma brucei gambiense

    Mol Biochem Parasitol

    (1988)
  • T.N. Darling et al.

    Carbon dioxide abolishes the reverse Pasteur effect in Leishmania major promastigotes

    Mol Biochem Parasitol

    (1989)
  • B.M. Bakker et al.

    Glycolysis in bloodstream form Trypanosoma brucei can be understood in terms of the kinetics of the glycolytic enzymes

    J Biol Chem

    (1997)
  • M.A. Albert et al.

    Experimental and in silico analyses of glycolytic flux control in bloodstream form Trypanosoma brucei

    J Biol Chem

    (2005)
  • P.A.M. Michels et al.

    Metabolic aspects of glycosomes in Trypanosomatidae-new data and views

    Parasitol Today

    (2000)
  • F.R. Opperdoes et al.

    In silico prediction of the glycosomal enzymes of Leishmania major and trypanosomes

    Mol Biochem Parasitol

    (2006)
  • N. Visser et al.

    Glycolysis in Trypanosoma brucei

    Eur J Biochem

    (1980)
  • C.W. Van Roermund et al.

    The membrane of peroxisomes in Saccharomyces cerevisiae is impermeable to NAD(H) and acetyl-CoA under in vivo conditions

    EMBO J

    (1995)
  • R.J.A. Wanders et al.

    Lipid metabolism in relation to human disease. Chapter 7, Permeability properties of peroxisomes

    Molec Aspects Med

    (1998)
  • G.F. Sprague et al.

    Isolation and characterization of Saccharomyces cerevisiae mutants defective in glycerol catabolism

    J Bacteriol

    (1977)
  • Cited by (0)

    1

    Present address: Department of Microbiology, Universidad Peruana Cayetano Heredia Av. Honorio Delgado 430, Lima 31, A.P. 4314, Perú.

    View full text