Katrin Henze

Biochemistry and evolution of anaerobic energy metabolism in eukaryotes.

SUMMARY Major insights into the phylogenetic distribution, biochemistry, and evolutionary significance of organelles involved in ATP synthesis (energy metabolism) in eukaryotes that thrive in anaerobic environments for all or part of their life cycles have accrued in recent years. All known eukaryotic groups possess an organelle of mitochondrial origin, mapping the origin of mitochondria to the eukaryotic common ancestor, and genome sequence data are rapidly accumulating for eukaryotes that possess anaerobic mitochondria, hydrogenosomes, or mitosomes. Here we review the available biochemical data on the enzymes and pathways that eukaryotes use in anaerobic energy metabolism and summarize the metabolic end products that they generate in their anaerobic habitats, focusing on the biochemical roles that their mitochondria play in anaerobic ATP synthesis. We present metabolic maps of compartmentalized energy metabolism for 16 well-studied species. There are currently no enzymes of core anaerobic energy metabolism that are specific to any of the six eukaryotic supergroup lineages; genes present in one supergroup are also found in at least one other supergroup. The gene distribution across lineages thus reflects the presence of anaerobic energy metabolism in the eukaryote common ancestor and differential loss during the specialization of some lineages to oxic niches, just as oxphos capabilities have been differentially lost in specialization to anoxic niches and the parasitic life-style. Some facultative anaerobes have retained both aerobic and anaerobic pathways. Diversified eukaryotic lineages have retained the same enzymes of anaerobic ATP synthesis, in line with geochemical data indicating low environmental oxygen levels while eukaryotes arose and diversified.

An overview of endosymbiotic models for the origins of eukaryotes, their ATP-producing organelles (mitochondria and hydrogenosomes), and their heterotrophic lifestyle.

Abstract The evolutionary processes underlying the differentness of prokaryotic and eukaryotic cells and the origin of the latters organelles are still poorly understood. For about 100 years, the principle of endosymbiosis has figured into thoughts as to how these processes might have occurred. A number of models that have been discussed in the literature and that are designed to explain this difference are summarized. The evolutionary histories of the enzymes of anaerobic energy metabolism (oxygenindependent ATP synthesis) in the three basic types of heterotrophic eukaryotes those that lack organelles of ATP synthesis, those that possess mitochondria and those that possess hydrogenosomes play an important role in this issue. Traditional endosymbiotic models generally do not address the origin of the heterotrophic lifestyle and anaerobic energy metabolism in eukaryotes. Rather they take it as a given, a direct inheritance from the host that acquired mitochondria. Traditional models are contrasted to an alternative endosymbiotic model (the hydrogen hypothesis), which addresses the origin of heterotrophy and the origin of compartmentalized energy metabolism in eukaryotes.

How do mitochondrial genes get into the nucleus

It is well known that genes from chloroplasts and mitochondria were transferred to the nucleus many times during plant evolution. But in what form do the transferred genes physically make that intracellular journey--as RNA, as cDNA, as pieces of organelle DNA, or as whole organelle chromosomes? Current views focus upon cDNA as the vehicle, based upon some examples from plants. But other mechanisms, involving direct transfer of DNA from organelle chromosomes, could also account for the available data. Direct DNA transfer, rather than cDNA-mediated transfer, does occur today, and it probably prevailed during the early phases of organelle evolution.

HIGHER-PLANT CHLOROPLAST AND CYTOSOLIC FRUCTOSE-1,6-BISPHOPHOSPHATASE ISOENZYMES : ORIGINS VIA DUPLICATION RATHER THAN PROKARYOTE-EUKARYOTE DIVERGENCE

Full-size cDNAs encoding the precursors of chloroplast fructose-1,6-bisphosphatase (FBP), sedoheptulose-1,7-bisphosphatase (SBP), and the small subunit of Rubisco (RbcS) from spinach were cloned. These cDNAs complete the set of homologous probes for all nuclear-encoded enzymes of the Calvin cycle from spinach (Spinacia oleracea L.). FBP enzymes not only of higher plants but also of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a eubacterial origin of these eukaryotic nuclear genes. Chloroplast and cytosolic FBP isoenzymes of higher plants arose through a gene duplication event which occurred early in eukaryotic evolution. Both FBP and SBP of higher plant chloroplasts have acquired substrate specificity, i.e. have undergone functional specialization since their divergence from bifunctional FBP/SBP enzymes of free-living eubacteria.

Acetate:Succinate CoA-transferase in the Hydrogenosomes of Trichomonas vaginalis IDENTIFICATION AND CHARACTERIZATION

Acetate:succinate CoA-transferases (ASCT) are acetate-producing enzymes in hydrogenosomes, anaerobically functioning mitochondria and in the aerobically functioning mitochondria of trypanosomatids. Although acetate is produced in the hydrogenosomes of a number of anaerobic microbial eukaryotes such as Trichomonas vaginalis, no acetate producing enzyme has ever been identified in these organelles. Acetate production is the last unidentified enzymatic reaction of hydrogenosomal carbohydrate metabolism. We identified a gene encoding an enzyme for acetate production in the genome of the hydrogenosome-containing protozoan parasite T. vaginalis. This gene shows high similarity to Saccharomyces cerevisiae acetyl-CoA hydrolase and Clostridium kluyveri succinyl-CoA:CoA-transferase. Here we demonstrate that this protein is expressed and is present in the hydrogenosomes where it functions as the T. vaginalis acetate:succinate CoA-transferase (TvASCT). Heterologous expression of TvASCT in CHO cells resulted in the expression of an active ASCT. Furthermore, homologous overexpression of the TvASCT gene in T. vaginalis resulted in an equivalent increase in ASCT activity. It was shown that the CoA transferase activity is succinate-dependent. These results demonstrate that this acetyl-CoA hydrolase/transferase homolog functions as the hydrogenosomal ASCT of T. vaginalis. This is the first hydrogenosomal acetate-producing enzyme to be identified. Interestingly, TvASCT does not share any similarity with the mitochondrial ASCT from Trypanosoma brucei, the only other eukaryotic succinate-dependent acetyl-CoA-transferase identified so far. The trichomonad enzyme clearly belongs to a distinct class of acetate:succinate CoA-transferases. Apparently, two completely different enzymes for succinate-dependent acetate production have evolved independently in ATP-generating organelles.

Acetate formation in the energy metabolism of parasitic helminths and protists

Formation and excretion of acetate as a metabolic end product of energy metabolism occurs in many protist and helminth parasites, such as the parasitic helminths Fasciola hepatica, Haemonchus contortus and Ascaris suum, and the protist parasites, Giardia lamblia, Entamoeba histolytica, Trichomonas vaginalis as well as Trypanosoma and Leishmania spp. In all of these parasites acetate is a main end product of their energy metabolism, whereas acetate formation does not occur in their mammalian hosts. Acetate production might therefore harbour novel targets for the development of new anti-parasitic drugs. In parasites, acetate is produced from acetyl-CoA by two different reactions, both involving substrate level phosphorylation, that are catalysed by either a cytosolic acetyl-CoA synthetase (ACS) or an organellar acetate:succinate CoA-transferase (ASCT). The ACS reaction is directly coupled to ATP synthesis, whereas the ASCT reaction yields succinyl-CoA for ATP formation via succinyl-CoA synthetase (SCS). Based on recent work on the ASCTs of F. hepatica, T. vaginalis and Trypanosoma brucei we suggest the existence of three subfamilies of enzymes within the CoA-transferase family I. Enzymes of these three subfamilies catalyse the ASCT reaction in eukaryotes via the same mechanism, but the subfamilies share little sequence homology. The CoA-transferases of the three subfamilies are all present inside ATP-producing organelles of parasites, those of subfamily IA in the mitochondria of trypanosomatids, subfamily IB in the mitochondria of parasitic worms and subfamily IC in hydrogenosome-bearing parasites. Together with the recent characterisation among non-parasitic protists of yet a third route of acetate formation involving acetate kinase (ACK) and phosphotransacetylase (PTA) that was previously unknown among eukaryotes, these recent developments provide a good opportunity to have a closer look at eukaryotic acetate formation.

Early Cell Evolution, Eukaryotes, Anoxia, Sulfide, Oxygen, Fungi First (?), and a Tree of Genomes Revisited

Genomes contain evidence for the history of life and furthermore contain evidence for lateral gene transfer, which was an important part of that history. The geological record also contains evidence for the history of life, and newer findings indicates that the Earths oceans were largely anoxic and highly sulfidic up until about 0.6 billion years ago. Eukaryotes, which fossil data indicate to have been in existence for at least 1.5 billion years, must have therefore spent much of their evolutionary history in oxygen‐poor and sulfide‐rich environments. Many eukaryotes still inhabit such environments today. Among eukaryotes, organelles also contain evidence for the history of life and have preserved abundant traces of their anaerobic past in the form of energy metabolic pathways. New views of eukaryote phylogeny suggest that fungi may be among the earliest‐branching eukaryotes. From the standpoint of the fungal feeding habit (osmotrophy rather than phagotrophy) and from the standpoint of the diversity in their ATP‐producing pathways, a eukaryotic tree with fungi first would make sense. Because of lateral gene transfer and endosymbiosis, branches in the tree of genomes intermingle and occasionally fuse, but the overall contours of cell history nonetheless seem sketchable and roughly correlateable with geological time. IUBMB Life, 55: 193‐204, 2003

Origins of hydrogenosomes and mitochondria.

Complete genome sequences for many oxygen-respiring mitochondria, as well as for some bacteria, leave no doubt that mitochondria are descendants of alpha-proteobacteria, a finding for which the endosymbiont hypothesis can easily account. Yet a wealth of data indicate that mitochondria and hydrogenosomes - the ATP-producing organelles of many anaerobic protists - share a common ancestry, a finding that traditional formulations of the endosymbiont hypothesis less readily accommodates. Available evidence suggests that a more in-depth understanding of the origins of eukaryotes and their organelles will hinge upon data from the genomes of protists that synthesize ATP without the need for oxygen.

Bacterial-type oxygen detoxification and iron-sulfur cluster assembly in amoebal relict mitochondria.

The assembly of vital reactive iron‐sulfur (Fe‐S) cofactors in eukaryotes is mediated by proteins inherited from the original mitochondrial endosymbiont. Uniquely among eukaryotes, however, Entamoeba and Mastigamoeba lack such mitochondrial‐type Fe‐S cluster assembly proteins and possess instead an analogous bacterial‐type system acquired by lateral gene transfer. Here we demonstrate, using immunomicroscopy and biochemical methods, that beyond their predicted cytosolic distribution the bacterial‐type Fe‐S cluster assembly proteins NifS and NifU have been recruited to function within the relict mitochondrial organelles (mitosomes) of Entamoeba histolytica. Both Nif proteins are 10‐fold more concentrated within mitosomes compared with their cytosolic distribution suggesting that active Fe‐S protein maturation occurs in these organelles. Quantitative immunoelectron microscopy showed that amoebal mitosomes are minute but highly abundant cellular structures that occupy up to 2% of the total cell volume. In addition, protein colocalization studies allowed identification of the amoebal hydroperoxide detoxification enzyme rubrerythrin as a mitosomal protein. This protein contains functional Fe‐S centres and exhibits peroxidase activity in vitro. Our findings demonstrate the role of analogous protein replacement in mitochondrial organelle evolution and suggest that the relict mitochondrial organelles of Entamoeba are important sites of metabolic activity that function in Fe‐S protein‐mediated oxygen detoxification.

Euglena gracilis Rhodoquinone:Ubiquinone Ratio and Mitochondrial Proteome Differ under Aerobic and Anaerobic Conditions

Euglena gracilis cells grown under aerobic and anaerobic conditions were compared for their whole cell rhodoquinone and ubiquinone content and for major protein spots contained in isolated mitochondria as assayed by two-dimensional gel electrophoresis and mass spectrometry sequencing. Anaerobically grown cells had higher rhodoquinone levels than aerobically grown cells in agreement with earlier findings indicating the need for fumarate reductase activity in anaerobic wax ester fermentation in Euglena. Microsequencing revealed components of complex III and complex IV of the respiratory chain and the E1β subunit of pyruvate dehydrogenase to be present in mitochondria of aerobically grown cells but lacking in mitochondria from anaerobically grown cells. No proteins were identified as specific to mitochondria from anaerobically grown cells. cDNAs for the E1α, E2, and E3 subunits of mitochondrial pyruvate dehydrogenase were cloned and shown to be differentially expressed under aerobic and anaerobic conditions. Their expression patterns differed from that of mitochondrial pyruvate:NADP+ oxidoreductase, the N-terminal domain of which is pyruvate:ferredoxin oxidoreductase, an enzyme otherwise typical of hydrogenosomes, hydrogen-producing forms of mitochondria found among anaerobic protists. The Euglena mitochondrion is thus a long sought intermediate that unites biochemical properties of aerobic and anaerobic mitochondria and hydrogenosomes because it contains both pyruvate:ferredoxin oxidoreductase and rhodoquinone typical of hydrogenosomes and anaerobic mitochondria as well as pyruvate dehydrogenase and ubiquinone typical of aerobic mitochondria. Our data show that under aerobic conditions Euglena mitochondria are prepared for anaerobic function and furthermore suggest that the ancestor of mitochondria was a facultative anaerobe, segments of whose physiology have been preserved in the Euglena lineage.