Schematic map of pyruvate metabolism in the cytosol and the hydrogenosomes of metronidazole-sensitive and -resistant T. vaginalis and T. foetus . Enzymes marked with asterisks are demonstrated only in T. foetus . Steps depicted by dashed arrows were demonstrated only in metronidazole-resistant T. foetus strains. The major cytosolic end product in metronidazole-susceptible strains of T. vaginalis is lactate, and that in T. foetus is succinate. In metronidazole-resistant T. vaginalis or T. foetus , the major cytosolic end product is lactate or ethanol, respectively. LDH is not present in T. foetus . Acetate is produced only in hydrogenosomes of metronidazole-susceptible T. vaginalis and T. foetus . Abbreviations are as follows: MDH, malate dehydrogenase (decarboxylating, malate to pyruvate); LDH, lactate dehydrogenase; Fu, fumarase; FR, fumarate reductase; PDC, pyruvate decarboxylase; ADH, alcohol dehydrogenase; Fd-ox, oxidized ferredoxin; Fd-rd, reduced ferredoxin; Pi, inorganic phosphate. Steps indicated by numbers are catalyzed by the following enzymes: 1, pyruvate:ferredoxin oxidoreductase; 2, NADH-dependent malate dehydrogenase (decarboxylating); 3, NADH:ferredoxin oxidoreductase activity of 51-kDa and 24-kDa catalytic flavoprotein components of complex I; 4, ferredoxin-dependent Fe-hydrogenase; 5, hypothetical NADH-dependent 65-kDa Fe-hydrogenase; 6, acetate:succinate CoA- transferase; 7, succinate thiokinase. 

Contexts in source publication

Context 1

... histolytica lacks PK activity (255) and that pyruvate is synthesized either by PPDK or through the third pathway mediated by PEP carboxyphosphotransferase, malate dehydrogenase, and malic enzyme, a recent report demonstrated the presence of PK activity and a putative PK gene in E. histolytica (269). No PPDK activity has been detected in T. vaginalis (209), and pyruvate is synthesized pre- dominantly by PK or through the oxaloacetate/malate pathway. Although two PPDK genes were reported recently in T. vaginalis (288), functional characterization of these genes is lacking. The glycolytic pyrophosphate-dependent enzymes PPDK and phosphofructokinase are proposed targets for therapeutic intervention. These pyrophosphate-dependent enzymes are absent in the human host and can be inhibited with pyrophos- phate analogues such as bisphosphonates. In order to validate PPDK as a rational drug target, it is essential to understand which pathway plays the major role in pyruvate formation in these protozoan parasites. In particular, mechanisms that control the expression of individual pathways depending upon substrate availability, oxygen tension, and other environmental factors are not well understood. The conversion of pyruvate to acetyl coenzyme A (acetyl- CoA) is catalyzed by pyruvate:ferredoxin oxidoreductase (PFOR), which utilizes ferredoxin rather than NAD ϩ as an electron acceptor in E. histolytica (257), G. intestinalis (307, 308), and T. vaginalis (330) in place of the pyruvate dehydrogenase complex found in aerobic bacteria and eukaryotes. Pyruvate dehydrogenase in the mitochondria of aerobic eu- karyotes catalyzes an oxidative decarboxylation to form acetyl- CoA and NADH. Energy metabolism in these protists was recently reviewed in more detail (112, 217). The end products of energy metabolism in “amitochondriate” protozoa are influenced by the O 2 tension (7). In addition, the major fermentation end products differ remarkably among these three organisms. For instance, in G. intestinalis , which has alanine aminotransferase, alanine is the major product of carbohydrate metabolism under strict anaerobic conditions (Fig. 1b) (243, 244). However, ethanol production is stimulated under conditions of low oxygen ( Ͻ 0.25 ␮ M), while alanine production is suppressed; the major end products are ethanol and CO 2 (243). Under conditions of higher oxygen ( Ͼ 46 ␮ M), where alanine production is completely inhibited, the major products from acetyl-CoA are acetate and CO 2 . In contrast, the major end product in E. histolytica under anaerobic conditions is ethanol. A putative alanine aminotransferase gene is present in the E. histolytica genome, but its functional identity needs to be demonstrated (185). Under microaerophilic conditions, both ethanol and acetate are formed as end products (203, 325). In Trichomonas , where part of the energy metabolism is compartmentalized in the hydrogenosome, electrons produced by the oxidation of pyruvate catalyzed by PFOR are donated to an Fe-hydrogenase via ferredoxin, which produces hydrogen gas (H 2 ) by transferring electrons to hydrogen ions (Fig. 2). ATP is generated exclusively by substrate-level phosphoryla- tion in the hydrogenosomes (75). In wild-type T. vaginalis , the major end product from pyruvate is lactate produced by lactate dehydrogenase in the cytosol; lactate dehydrogenase is present only in T. vaginalis . The preferred end product also differs between Trichomonas species and between wild-type and drug- resistant strains (168) (see below). In metronidazole-susceptible T. foetus , a Trichomonas species that affects cattle, either succinate or ethanol is the major or minor metabolic end product, respectively, in the cytosol (168), because lactate dehydrogenase is absent in this organism. In the hydrogenosomes, acetate is the only end product of both metronidazole- susceptible T. vaginalis and T. foetus strains. In contrast, major end products in the cytosol of metronidazole-resistant T. vaginalis and T. foetus strains are lactate and ethanol, respectively. Recently the NADH dehydrogenase module (also called NADH:ubiquinone oxidoreductase) of complex I was identified in T. vaginalis (75, 140, 141). Similar to mitochondrial respiratory complex I, NADH dehydrogenase can reduce a variety of electron carriers, including ubiquinone. Unlike the mitochondrial enzyme, ferredoxin is used as an electron carrier for hydrogen production in a reaction catalyzed by ferredoxin- dependent Fe-hydrogenase. Malate is one of the major hydrogenosomal substrates and is oxidatively decarboxylated to form pyruvate and CO 2 by a NAD-dependent malic enzyme. The electrons are transferred from NADH to ferredoxin by an NADH dehydrogenase homologous to the catalytic module of mitochondrial complex I. Thus, the discovery of an NADH dehydrogenase module of complex I solved the long-lasting conundrum of how Trichomonas regenerates NAD ϩ , which is essential for malate oxidation in the hydrogenosome (Fig. 2). It was previously shown that T. vaginalis possesses two additional 2-keto acid oxidoreductases besides PFOR for energy production (34). These two 2-keto acid oxidoreductases, KOR1 and KOR2, prefer indolepyruvate as a substrate. KOR1 is present in both metronidazole-sensitive and -resistant strains, while KOR2 is present only in metronidazole-resistant strains of T. vaginalis (34, 73, 313). It was reported that 2-keto acid oxidoreductase activity increased in the metronidazole- resistant strain (34). However, neither KOR1 nor KOR2 could donate electrons to ferredoxin in the metronidazole-resistant line, because no detectable ferredoxin was produced in this strain (73) (see below). Pyruvate:ferredoxin oxidoreductase. Eukaryotic PFOR is a homodimeric protein with a molecular mass of ϳ 200 to 300 kDa containing 2[4Fe-4S] clusters and thiamine phosphate. Its structure is similar to that of PFORs from a wide range of eubacteria (138); e.g., PFOR from the eubacterium Clostridium acetobutylicum is a homodimer of 123-kDa subunits (207). PFOR from the thermophilic archaebacterium Pyrococcus furiosus is composed of four fragmented subunits (29, 161), all of which share significant homology to PFORs from eubacteria, fungi, and protists (138). PFORs from E. histolytica , G. intestinalis , and T. vaginalis are homodimers of 240 to 280 kDa containing 2[4Fe-4S] clusters and thiamine phosphate (257, 264, 308, 330). Despite the similar overall structure and the Fe-S clusters of PFORs in these “amitochondriate” protists, the intracellular localization of PFOR is divergent. While PFORs from both archaea and eubacteria are cytosolic, biochemical characterization (179, 330) has established that PFOR is localized to hydrogenosomes and is associated with the hydrogenosome membrane in T. vaginalis (330). In addition, a 120-kDa surface glycoprotein that shares cross-reacting epitopes with PFOR is involved in the adhesion of T. vaginalis trophozoites (213). This may suggest that PFOR or a PFOR- related protein is associated with hydrogenosomes and the plasma membrane, but this premise is still a matter of debate and needs to be independently verified. The PFORs from both G. intestinalis (81, 308) and E. histolytica (263, 274) were also suggested to be associated with the plasma membrane. PFOR was shown to be associated with the plasma membrane and with a cytoplasmic structure in E. histolytica that appeared as a ring form or a compact small body (263). However, the ma- jority of PFOR activity was detected in an 80,000 ϫ g super- natant fraction (257), suggesting that the proposed membrane association of E. histolytica PFOR (263, 274) might be tran- sient and/or weak. At the primary sequence level, T. vaginalis PFOR possesses the putative mitochondrial targeting peptide at the amino terminus (142). In contrast, neither E. histolytica nor G. intestinalis PFOR possesses the mitochondrial targeting sequence. Since pyruvate metabolism is not compartmentalized in either of these organisms like it is in T. vaginalis , the physiological significance of the possible membrane association of PFOR in G. intestinalis and E. histolytica is not well understood. Ferredoxin. Ferredoxin is another important class of proteins containing Fe-S clusters. Ferredoxins in the three microaerophilic/anaerobic parasitic protists differ in the nature of the Fe-S clusters they contain and their intracellular localization, i.e., cytosolic or hydrogenosomal. The E. histolytica genome encodes at least three ferredoxins (185), all of which contain either 2[4Fe-4S] clusters or [4Fe-4S] and [3Fe-4S] clusters, which correspond to a molecular mass of ϳ 6 kDa, as previously characterized (256). E. histolytica apparently lacks [2Fe-2S] ferredoxin (15, 17), but ferredoxins containing these clusters are present in T. vaginalis and G. intestinalis . A larger ferredoxin from Trichomonas with one [2Fe-2S] cluster corresponding to a molecular mass of ϳ 10 kDa was biochemically characterized and was localized in hydrogenosomes (116). The genome of G. intestinalis encodes at least four ferredoxins: one ferredoxin containing one [2Fe-2S] cluster (ferredoxin I) and three ferredoxins containing either 2[4Fe-4S] clusters or one [4Fe-4S] cluster and one [3Fe-4S] cluster (ferredoxins 1 to 3) (229). Among these ferredoxins, only ferredoxin I was shown to accept electrons from PFOR in vitro (308). The physiological role of [4Fe-4S] ferredoxins in G. intestinalis remains unclear. The small 2[4Fe-4S] ferredoxin of E. histolytica is evolu- tionarily closest to ferredoxin from anaerobic bacteria (143), while the larger [2Fe-2S] ferredoxin of Trichomonas hydrogenosomes shows a close kinship to ferredoxins of the cyto- chrome P450-linked mixed-function oxidase systems of bacterial and vertebrate mitochondria (154). In addition, G. intestinalis possesses two genes, and E. histolytica possesses one gene, encoding a putative ferredoxin nitroreductase which consists of an ...

Context 2

... specialized mitochondrion-related organelle or “hydrogeno- some” in T. vaginalis has compartmentalized pyruvate oxidation (168, 179, 215, 216, 217, 218, 219) (Fig. 2). Besides compartmentalized energy metabolism in the T. vaginalis hydrogenosome, functional differences exist in the mitochondrion-related organelles among these three organisms. It was recently demonstrated that the ISC pathway, involved in iron-sulfur (Fe-S) cluster formation, is localized in the mitosomes of G. intestinalis and the hydrogenosomes of T. vaginalis (294, 304). In contrast, the E. histolytica mitosome lacks the ISC system. Instead, E. histolytica possesses a nitrogen fixation (NIF)-like system localized in the cytosol that is similar to that found in enteric Epsilonprotobacteria (15; V. Ali et al., unpublished data). Fe-S cluster biosynthesis is further described in “Physiological Importance of Cysteine and Fe-S Cluster Biosynthesis” below. In Entamoeba , Giardia , and Trichomonas , glucose is not oxidized to CO 2 and H 2 O as it is in aerobic metabolism but is instead catabolized to acetate, succinate, ethanol, alanine, and CO 2 . The predominant metabolic end products and catabolic pathways depend on the organism and its environmental and physiological (e.g., drug sensitivity) states (see below). The pathways involved in the biosynthesis of pyruvate from phos- phoenolpyruvate (PEP) diverge among these organisms (Fig. 1a). G. intestinalis can utilize three pathways to synthesize pyruvate: through pyruvate kinase (PK), pyruvate phosphate dikinase (PPDK), and a pathway mediated by PEP carboxyphosphotransferase, malate dehydrogenase (oxaloacetate to malate), and malic enzyme (decarboxylating, malate to pyruvate). It was assumed that, in G. intestinalis , the conversion of PEP to pyruvate operated mainly through PPDK (7). Although it was reported earlier that E. histolytica lacks PK activity (255) and that pyruvate is synthesized either by PPDK or through the third pathway mediated by PEP carboxyphosphotransferase, malate dehydrogenase, and malic enzyme, a recent report demonstrated the presence of PK activity and a putative PK gene in E. histolytica (269). No PPDK activity has been detected in T. vaginalis (209), and pyruvate is synthesized pre- dominantly by PK or through the oxaloacetate/malate pathway. Although two PPDK genes were reported recently in T. vaginalis (288), functional characterization of these genes is lacking. The glycolytic pyrophosphate-dependent enzymes PPDK and phosphofructokinase are proposed targets for therapeutic intervention. These pyrophosphate-dependent enzymes are absent in the human host and can be inhibited with pyrophos- phate analogues such as bisphosphonates. In order to validate PPDK as a rational drug target, it is essential to understand which pathway plays the major role in pyruvate formation in these protozoan parasites. In particular, mechanisms that control the expression of individual pathways depending upon substrate availability, oxygen tension, and other environmental factors are not well understood. The conversion of pyruvate to acetyl coenzyme A (acetyl- CoA) is catalyzed by pyruvate:ferredoxin oxidoreductase (PFOR), which utilizes ferredoxin rather than NAD ϩ as an electron acceptor in E. histolytica (257), G. intestinalis (307, 308), and T. vaginalis (330) in place of the pyruvate dehydrogenase complex found in aerobic bacteria and eukaryotes. Pyruvate dehydrogenase in the mitochondria of aerobic eu- karyotes catalyzes an oxidative decarboxylation to form acetyl- CoA and NADH. Energy metabolism in these protists was recently reviewed in more detail (112, 217). The end products of energy metabolism in “amitochondriate” protozoa are influenced by the O 2 tension (7). In addition, the major fermentation end products differ remarkably among these three organisms. For instance, in G. intestinalis , which has alanine aminotransferase, alanine is the major product of carbohydrate metabolism under strict anaerobic conditions (Fig. 1b) (243, 244). However, ethanol production is stimulated under conditions of low oxygen ( Ͻ 0.25 ␮ M), while alanine production is suppressed; the major end products are ethanol and CO 2 (243). Under conditions of higher oxygen ( Ͼ 46 ␮ M), where alanine production is completely inhibited, the major products from acetyl-CoA are acetate and CO 2 . In contrast, the major end product in E. histolytica under anaerobic conditions is ethanol. A putative alanine aminotransferase gene is present in the E. histolytica genome, but its functional identity needs to be demonstrated (185). Under microaerophilic conditions, both ethanol and acetate are formed as end products (203, 325). In Trichomonas , where part of the energy metabolism is compartmentalized in the hydrogenosome, electrons produced by the oxidation of pyruvate catalyzed by PFOR are donated to an Fe-hydrogenase via ferredoxin, which produces hydrogen gas (H 2 ) by transferring electrons to hydrogen ions (Fig. 2). ATP is generated exclusively by substrate-level phosphoryla- tion in the hydrogenosomes (75). In wild-type T. vaginalis , the major end product from pyruvate is lactate produced by lactate dehydrogenase in the cytosol; lactate dehydrogenase is present only in T. vaginalis . The preferred end product also differs between Trichomonas species and between wild-type and drug- resistant strains (168) (see below). In metronidazole-susceptible T. foetus , a Trichomonas species that affects cattle, either succinate or ethanol is the major or minor metabolic end product, respectively, in the cytosol (168), because lactate dehydrogenase is absent in this organism. In the hydrogenosomes, acetate is the only end product of both metronidazole- susceptible T. vaginalis and T. foetus strains. In contrast, major end products in the cytosol of metronidazole-resistant T. vaginalis and T. foetus strains are lactate and ethanol, respectively. Recently the NADH dehydrogenase module (also called NADH:ubiquinone oxidoreductase) of complex I was identified in T. vaginalis (75, 140, 141). Similar to mitochondrial respiratory complex I, NADH dehydrogenase can reduce a variety of electron carriers, including ubiquinone. Unlike the mitochondrial enzyme, ferredoxin is used as an electron carrier for hydrogen production in a reaction catalyzed by ferredoxin- dependent Fe-hydrogenase. Malate is one of the major hydrogenosomal substrates and is oxidatively decarboxylated to form pyruvate and CO 2 by a NAD-dependent malic enzyme. The electrons are transferred from NADH to ferredoxin by an NADH dehydrogenase homologous to the catalytic module of mitochondrial complex I. Thus, the discovery of an NADH dehydrogenase module of complex I solved the long-lasting conundrum of how Trichomonas regenerates NAD ϩ , which is essential for malate oxidation in the hydrogenosome (Fig. 2). It was previously shown that T. vaginalis possesses two additional 2-keto acid oxidoreductases besides PFOR for energy production (34). These two 2-keto acid oxidoreductases, KOR1 and KOR2, prefer indolepyruvate as a substrate. KOR1 is present in both metronidazole-sensitive and -resistant strains, while KOR2 is present only in metronidazole-resistant strains of T. vaginalis (34, 73, 313). It was reported that 2-keto acid oxidoreductase activity increased in the metronidazole- resistant strain (34). However, neither KOR1 nor KOR2 could donate electrons to ferredoxin in the metronidazole-resistant line, because no detectable ferredoxin was produced in this strain (73) (see below). Pyruvate:ferredoxin oxidoreductase. Eukaryotic PFOR is a homodimeric protein with a molecular mass of ϳ 200 to 300 kDa containing 2[4Fe-4S] clusters and thiamine phosphate. Its structure is similar to that of PFORs from a wide range of eubacteria (138); e.g., PFOR from the eubacterium Clostridium acetobutylicum is a homodimer of 123-kDa subunits (207). PFOR from the thermophilic archaebacterium Pyrococcus furiosus is composed of four fragmented subunits (29, 161), all of which share significant homology to PFORs from eubacteria, fungi, and protists (138). PFORs from E. histolytica , G. intestinalis , and T. vaginalis are homodimers of 240 to 280 kDa containing 2[4Fe-4S] clusters and thiamine phosphate (257, 264, 308, 330). Despite the similar overall structure and the Fe-S clusters of PFORs in these “amitochondriate” protists, the intracellular localization of PFOR is divergent. While PFORs from both archaea and eubacteria are cytosolic, biochemical characterization (179, 330) has established that PFOR is localized to hydrogenosomes and is associated with the hydrogenosome membrane in T. vaginalis (330). In addition, a 120-kDa surface glycoprotein that shares cross-reacting epitopes with PFOR is involved in the adhesion of T. vaginalis trophozoites (213). This may suggest that PFOR or a PFOR- related protein is associated with hydrogenosomes and the plasma membrane, but this premise is still a matter of debate and needs to be independently verified. The PFORs from both G. intestinalis (81, 308) and E. histolytica (263, 274) were also suggested to be associated with the plasma membrane. PFOR was shown to be associated with the plasma membrane and with a cytoplasmic structure in E. histolytica that appeared as a ring form or a compact small body (263). However, the ma- jority of PFOR activity was detected in an 80,000 ϫ g super- natant fraction (257), suggesting that the proposed membrane association of E. histolytica PFOR (263, 274) might be tran- sient and/or weak. At the primary sequence level, T. vaginalis PFOR possesses the putative mitochondrial targeting peptide at the amino terminus (142). In contrast, neither E. histolytica nor G. intestinalis PFOR possesses the mitochondrial targeting sequence. Since pyruvate metabolism is not compartmentalized in either of these organisms like it is in T. vaginalis , the physiological significance of the possible membrane association of ...

Context 3

... e.g., pear or oval shaped, but the parasite takes on a more amoeboid appearance when attached to vaginal epithelial cells (19, 124). The trophozoite has four free anterior flagella [9(2) ϩ 2 arrangement] and a single recurrent flagellum incorporated into an undulating membrane which is supported by a noncontractile costa (282). The trophozoite divides by binary fission (247). T. vaginalis infection in women ranges from an asymptomatic carrier state to profound acute inflammatory disease (261). The parasite generally infects the squamous epithelium of the genital tract, but it is occasionally recovered from the urethra, fallopian tubes, and pelvis (117, 247). The incubation period is 4 to 28 days in about 50% of infected individuals (247). The clinical picture in acute infection includes vaginal discharge, odor, and edema or erythema. The discharge is classically described as frothy, but actually it is frothy in only 10% of patients (282). Small punctuate hemorrhagic spots may be found on the vaginal and cervical mucosae in 2 to 3% of patients. These signs and symptoms are cyclic and worsen around the time of menses. Other complications associated with trichomoniasis include adnexitis, pyosalpinx, and endome- tritis (260); infertility and low birth weight (123); and cervical erosion (204). In males, T. vaginalis infection is often asymptomatic but occasionally causes urethritis and prostatitis (166). Urogenital trichomoniasis in men is categorized into three groups: an asymptomatic carrier state identified through an investigation of the sexual contacts of infected women, acute trichomoniasis characterized by profuse purulent urethritis, and mild symptomatic disease which is clinically indistinguish- able from other causes of nongonococcal urethritis. The dura- tion of infection is 10 days or less in most male patients. In symptomatic men, common complaints include scanty clear to mucopurulent discharge, dysuria, and mild pruritus or a burn- ing sensation immediately after sexual intercourse (166). Com- plications associated with trichomoniasis include nongonococcal urethritis, prostatitis, balanoposthitis, urethral disease, and infertility (131, 167, 197). Pneumonia, bronchitis, and oral infection caused by T. vaginalis have also been documented (132). In rare cases, respiratory infections are acquired peri- natally from infected mothers (132, 158). In children, T. vaginalis can infect the urinary tract as well as the vagina. It has been suggested that T. vaginalis infection is associated with sterility, but there is no unequivocal report available. In contrast, there is an established causal relationship between T. vaginalis infection and adverse pregnancy outcomes (9). Similarly, T. vaginalis infection was associated with low birth weight and preterm delivery in 40% of black women (57, 279). In that study, in which 14,000 American women were examined, Trichomonas was also associated with high infant mortality. In cattle, Trichomonas foetus infection causes infertility, which results in a tremendous economic loss (55, 133). It was suggested that T. vaginalis infection, as well as other STDs, increases susceptibility to HIV infection due to local inflammation and microscopic breaches in mucosal barriers (282). It was also suggested that T. vaginalis infection predisposes HIV carriers to symptomatic AIDS (211, 292). However, this issue is still debatable. Conversely, immunosuppression from HIV infection increases susceptibility to STDs (173, 211, 250, 282, 283, 292). Most higher eukaryotic organisms, including mammals, depend primarily on aerobic metabolism for their energy production. In contrast, certain anaerobic/microaerophilic eukaryotes, including Entamoeba , Giardia , and Trichomonas , lack typical and morpho- logically discernible mitochondria and the cytochrome-mediated oxidative phosphorylation found in aerobic organisms (217). Accordingly, these three parasites are often misleadingly referred to as “amitochondriate” protists. However, it is now well established that these organisms possess mitochondrion-related organelles, often with reductive or sometimes divergent functions (49, 104, 179, 194, 303, 304). For detailed discussions of the phylogenetic and biochemical aspects of the mitochondrion-related organelles, consult references 85, 86, 87, and 316. For the most recent reviews on the evolution of “amitochondriate” parasites, see references 84 and 156. These “amitochondriate” protists rely on substrate-level phosphorylation during glycolysis for ATP generation and mostly on fermentative metabolism for energy production (Fig. 1a and b). While the vestigial organelles or “mitosomes” (or “crypton” in E. histolytica ) in E. histolytica and G. intestinalis retain only resid- ual mitochondrial functions (49, 104, 194, 303, 304), the highly specialized mitochondrion-related organelle or “hydrogeno- some” in T. vaginalis has compartmentalized pyruvate oxidation (168, 179, 215, 216, 217, 218, 219) (Fig. 2). Besides compartmentalized energy metabolism in the T. vaginalis hydrogenosome, functional differences exist in the mitochondrion-related organelles among these three organisms. It was recently demonstrated that the ISC pathway, involved in iron-sulfur (Fe-S) cluster formation, is localized in the mitosomes of G. intestinalis and the hydrogenosomes of T. vaginalis (294, 304). In contrast, the E. histolytica mitosome lacks the ISC system. Instead, E. histolytica possesses a nitrogen fixation (NIF)-like system localized in the cytosol that is similar to that found in enteric Epsilonprotobacteria (15; V. Ali et al., unpublished data). Fe-S cluster biosynthesis is further described in “Physiological Importance of Cysteine and Fe-S Cluster Biosynthesis” below. In Entamoeba , Giardia , and Trichomonas , glucose is not oxidized to CO 2 and H 2 O as it is in aerobic metabolism but is instead catabolized to acetate, succinate, ethanol, alanine, and CO 2 . The predominant metabolic end products and catabolic pathways depend on the organism and its environmental and physiological (e.g., drug sensitivity) states (see below). The pathways involved in the biosynthesis of pyruvate from phos- phoenolpyruvate (PEP) diverge among these organisms (Fig. 1a). G. intestinalis can utilize three pathways to synthesize pyruvate: through pyruvate kinase (PK), pyruvate phosphate dikinase (PPDK), and a pathway mediated by PEP carboxyphosphotransferase, malate dehydrogenase (oxaloacetate to malate), and malic enzyme (decarboxylating, malate to pyruvate). It was assumed that, in G. intestinalis , the conversion of PEP to pyruvate operated mainly through PPDK (7). Although it was reported earlier that E. histolytica lacks PK activity (255) and that pyruvate is synthesized either by PPDK or through the third pathway mediated by PEP carboxyphosphotransferase, malate dehydrogenase, and malic enzyme, a recent report demonstrated the presence of PK activity and a putative PK gene in E. histolytica (269). No PPDK activity has been detected in T. vaginalis (209), and pyruvate is synthesized pre- dominantly by PK or through the oxaloacetate/malate pathway. Although two PPDK genes were reported recently in T. vaginalis (288), functional characterization of these genes is lacking. The glycolytic pyrophosphate-dependent enzymes PPDK and phosphofructokinase are proposed targets for therapeutic intervention. These pyrophosphate-dependent enzymes are absent in the human host and can be inhibited with pyrophos- phate analogues such as bisphosphonates. In order to validate PPDK as a rational drug target, it is essential to understand which pathway plays the major role in pyruvate formation in these protozoan parasites. In particular, mechanisms that control the expression of individual pathways depending upon substrate availability, oxygen tension, and other environmental factors are not well understood. The conversion of pyruvate to acetyl coenzyme A (acetyl- CoA) is catalyzed by pyruvate:ferredoxin oxidoreductase (PFOR), which utilizes ferredoxin rather than NAD ϩ as an electron acceptor in E. histolytica (257), G. intestinalis (307, 308), and T. vaginalis (330) in place of the pyruvate dehydrogenase complex found in aerobic bacteria and eukaryotes. Pyruvate dehydrogenase in the mitochondria of aerobic eu- karyotes catalyzes an oxidative decarboxylation to form acetyl- CoA and NADH. Energy metabolism in these protists was recently reviewed in more detail (112, 217). The end products of energy metabolism in “amitochondriate” protozoa are influenced by the O 2 tension (7). In addition, the major fermentation end products differ remarkably among these three organisms. For instance, in G. intestinalis , which has alanine aminotransferase, alanine is the major product of carbohydrate metabolism under strict anaerobic conditions (Fig. 1b) (243, 244). However, ethanol production is stimulated under conditions of low oxygen ( Ͻ 0.25 ␮ M), while alanine production is suppressed; the major end products are ethanol and CO 2 (243). Under conditions of higher oxygen ( Ͼ 46 ␮ M), where alanine production is completely inhibited, the major products from acetyl-CoA are acetate and CO 2 . In contrast, the major end product in E. histolytica under anaerobic conditions is ethanol. A putative alanine aminotransferase gene is present in the E. histolytica genome, but its functional identity needs to be demonstrated (185). Under microaerophilic conditions, both ethanol and acetate are formed as end products (203, 325). In Trichomonas , where part of the energy metabolism is compartmentalized in the hydrogenosome, electrons produced by the oxidation of pyruvate catalyzed by PFOR are donated to an Fe-hydrogenase via ferredoxin, which produces hydrogen gas (H 2 ) by transferring electrons to hydrogen ions (Fig. 2). ATP is generated exclusively by substrate-level phosphoryla- tion in the hydrogenosomes (75). In wild-type T. vaginalis , the major end product from ...