Proteins of the Glycine Decarboxylase Complex in the Hydrogenosome ofTrichomonas vaginalis

Abstract

Trichomonas vaginalis is a unicellular eukaryote that lacks mitochondria and contains a specialized organelle, the hydrogenosome, involved in carbohydrate metabolism and iron-sulfur cluster assembly. We report the identification of two glycine cleavage H proteins and a dihydrolipoamide dehydrogenase (L protein) of the glycine decarboxylase complex in T. vaginalis with predicted N-terminal hydrogenosomal presequences. Immunofluorescence analyses reveal that both H and L proteins are localized in hydrogenosomes, providing the first evidence for amino acid metabolism in this organelle. All three proteins were expressed in Escherichia coli and purified to homogeneity. The experimental K m of L protein for the two H proteins were 2.6 M and 3.7 M, consistent with both H proteins serving as substrates of L protein. Analyses using purified hydrogenosomes showed that endogenous H proteins exist as monomers and endogenous L protein as a homodimer in their native states. Phylogenetic analyses of L proteins revealed that the T. vaginalis homologue shares a common ancestry with dihydrolipoamide dehydrogenases from the firmicute bacteria, indicating its acquisition via a horizontal gene transfer event independent of the origins of mitochondria and hydrogenosomes.Hydrogenosomes are double membrane-bound organelles involved in iron-sulfur cluster assembly that produce molecular hydrogen and ATP via carbohydrate metabolism (30,40,41). These organelles were originally discovered in trichomonads and subsequently were detected in a variety of unrelated organisms, such as certain ciliates and fungi. Although the evolutionary origin(s) of hydrogenosomes is under debate, they appear to be polyphyletic, with ciliate hydrogenosomes being derived directly from mitochondria (3) and the origin of those in trichomonads and fungi less clear (11,17). Functional and phylogenetic data do, however, indicate that all hydrogenosomes are related to mitochondria (5,14,34,36,41).Trichomonas vaginalis is a deep-branching, microaerophilic, unicellular protist that lacks mitochondria and contains hydrogenosomes (30, 39). The lack of a genome in the hydrogenosomes of trichomonads precludes reconstruction of their ancestry directly as has been convincingly done for mitochondria (18). However, the phylogeny of genes that encode hydrogenosomal proteins, the hydrogenosomal targeting signals, and protein import machinery can provide indirect evidence for the evolutionary history of hydrogenosomes (4, 12, 43). Regardless of whether a protomitochondrion was modified to become a hydrogenosome or was lost during the adaptation of Trichomonas to anoxic environments, the absence of the tricarboxylic acid (TCA) cycle and many metabolic enzymes underscore the independent evolution of the T. vaginalis hydrogenosome as a unique organelle.Carbohydrates are the preferred source of nutrients for T. vaginalis; however, under conditions where carbohydrates are limiting, amino acids have been shown to sustain trichomonad growth and survival. Evidence of uptake of amino acids...

Lipoic Acid Metabolism in Microbial Pathogens

“…As is the case with G. lamblia (230) and E. histolytica (189), T. vaginalis relies on a pyruvate: ferredoxin oxidoreductase to produce acetyl-CoA (89,252). T. vaginalis diverges from other amitochondriate, anaerobic protozoans, however, in that it contains an L protein and two H protein paralogs, a feature that is so far unique to T. vaginalis (Table 6) (150). Both H proteins can be lipoylated, and both can serve as substrates of the L protein, indicating that they can participate in redox reactions involving protein-bound lipoate (150).…”

“…This situation is similar to that found in certain Firmicutes bacteria, such as Enterococcus faecalis, which also appear to lack P and T proteins (Table 6). Interestingly, the L protein found in T. vaginalis is most closely related to those of the Firmicutes and appears to have been acquired by horizontal gene transfer rather than through a mitochondrial precursor (150).…”

“…Both H proteins can be lipoylated, and both can serve as substrates of the L protein, indicating that they can participate in redox reactions involving protein-bound lipoate (150). The L protein is a dimeric dihydrolipoamide dehydrogenase with homology to E3 subunits and L proteins from other species (150). The P and T proteins have not been identified in T. vaginalis, suggesting either that they are highly divergent or that the parasite does not contain these proteins.…”

“…T. vaginalis diverges from other amitochondriate, anaerobic protozoans, however, in that it contains an L protein and two H protein paralogs, a feature that is so far unique to T. vaginalis (Table 6) (150). Both H proteins can be lipoylated, and both can serve as substrates of the L protein, indicating that they can participate in redox reactions involving protein-bound lipoate (150). The L protein is a dimeric dihydrolipoamide dehydrogenase with homology to E3 subunits and L proteins from other species (150).…”

“…When the GCV favors glycine biosynthesis, glycine can be used for protein translation or as a substrate of ␦-aminolevulinic acid synthase in the first step of heme synthesis (81). In eukaryotes, including plants, the GCV has been found to be strictly mitochondrial (39), except in the amitochondriate protozoan Trichomonas vaginalis, where components of the GCV are found in organelles related to mitochondria called hydrogenosomes (150).…”