Succinate Secreted by Trypanosoma brucei Is Produced by a Novel and Unique Glycosomal Enzyme, NADH-dependent Fumarate Reductase

Abstract

In all trypanosomatids, including Trypanosoma brucei, glycolysis takes place in peroxisome-like organelles called glycosomes. These are closed compartments wherein the energy and redox (NAD(+)/NADH) balances need to be maintained. We have characterized a T. brucei gene called FRDg encoding a protein 35% identical to Saccharomyces cerevisiae fumarate reductases. Microsequencing of FRDg purified from glycosome preparations, immunofluorescence, and Western blot analyses clearly identified this enzyme as a glycosomal protein that is only expressed in the procyclic form of T. brucei but is present in all the other trypanosomatids studied, i.e. Trypanosoma congolense, Crithidia fasciculata and Leishmania amazonensis. The specific inactivation of FRDg gene expression by RNA interference showed that FRDg is responsible for the NADH-dependent fumarate reductase activity detected in glycosomal fractions and that at least 60% of the succinate secreted by the T. brucei procyclic form (in the presence of d-glucose as the sole carbon source) is produced in the glycosome by FRDg. We conclude that FRDg plays a key role in the energy metabolism by participating in the maintenance of the glycosomal NAD(+)/NADH balance. We have also detected a significant pyruvate kinase activity in the cytosol of the T. brucei procyclic cells that was not observed previously. Consequently, we propose a revised model of glucose metabolism in procyclic trypanosomes that may also be valid for all other trypanosomatids except the T. brucei bloodstream form. Interestingly, H. Gest has hypothesized previously (Gest, H. (1980) FEMS Microbiol. Lett. 7, 73-77) that a soluble NADH-dependent fumarate reductase has been present in primitive organisms and evolved into the present day fumarate reductases, which are quinol-dependent. FRDg may have the characteristics of such an ancestral enzyme and is the only NADH-dependent fumarate reductase characterized to date.

Fumarate Is an Essential Intermediary Metabolite Produced by the Procyclic Trypanosoma brucei

“…The accession numbers of the various sequences used in this study are as follows: 1) ZP_00417946; 2) YP_261882; 3) AAM36330; 4) NP_890615; 5) CAH37820; 6) AAF41965; 7) NP_717819; 8) ZP_00752568; 9) YP_066688; 10) (15) was performed by expression of stem-loop "sense/antisense" molecules of the targeted sequences (38) introduced in the pLew100 expression vector (kindly provided by E. Wirtz and G. Cross) (39). Construction of the pLew-FHc-SAS and pLewFHm-SAS plasmids, which target the FHm gene (from position 1211 to 1668 bp) and the FHc gene (from position 1114 to 1605 bp), respectively, was performed as described (25,28,30,32,38). Briefly, PCR-amplified 531-or 588-bp fragments containing the antisense FHc or FHm sequences (458 or 492 bp of targeted sequence, plus 40 or 72 bp used as a spacer to form the loop between the annealing sense and antisense sequences, respectively) and the restriction sites added to the primers were inserted in the HindIII and BamHI restriction sites of the pLew100 plasmid.…”

“…Subcellular Localization of FHm and FHc-In the procyclic form of T. brucei, fumarases convert malate into fumarate, the later being the substrate of the glycosomal and mitochondrial fumarate reductases (FRDg and FRDm1) to produce the excreted succinate (30,32). Consequently, it is tempting to propose that these two T. brucei fumarase isoforms are glycosomal and mitochondrial enzymes.…”

“…Malate is then converted into succinate by fumarases and NADH-dependent fumarate reductases (FRD). Approximately two-thirds of the succinate is produced in the glycosomes by a glycosomal FRD isoform (FRDg) (30), whereas a mitochondrial FRD isoform (FRDm1) produces the remaining succinate (32). This model would also predict that malate is exchanged between the glycosomal and mitochondrial compartments, and fumarase isoforms are present in both compartments to provide both FRD isoforms with fumarate (32).…”

“…Instead, acetyl-CoA is converted into acetate (28,29), which represents 20 -40% of the excreted end products from the glucose metabolism by the procyclic trypanosomes. According to the current model, succinate is the main excreted end product (60 -70%) (27,30); it is produced in both the glycosomes and the mitochondrion, by the so-called succinic fermentation pathway (21, 22, 30 -32). About half of the phosphoenolpyruvate (PEP) produced in the cytosol re-enters the glycosomes to be converted into malate by PEP carboxykinase and malate dehydrogenase.…”

NADH desidrogenase mitocondrial de Trypanosoma cruzi: subunidade 7 para diagnóstico diferencial de isolados humanos e análise funcional.

“…Por outro lado, muitos dos processos elucidados nos tripanossomas africanos são compartilhados por outras espécies de tripanossomatídeos (BESTEIRO et al, 2002).…”