Pyruvate:NADP Oxidoreductase from the Mitochondrion of Euglena gracilis and from the Apicomplexan Cryptosporidium parvum: A Biochemical Relic Linking Pyruvate Metabolism in Mitochondriate and Amitochondriate Protists

Abstract

Most eukaryotes perform the oxidative decarboxylation of pyruvate in mitochondria using pyruvate dehydrogenase (PDH). Eukaryotes that lack mitochondria also lack PDH, using instead the O(2)-sensitive enzyme pyruvate : ferredoxin oxidoreductase (PFO), which is localized either in the cytosol or in hydrogenosomes. The facultatively anaerobic mitochondria of the photosynthetic protist Euglena gracilis constitute a hitherto unique exception in that these mitochondria oxidize pyruvate with the O(2)-sensitive enzyme pyruvate : NADP oxidoreductase (PNO). Cloning and analysis of Euglena PNO revealed that the cDNA encodes a mitochondrial transit peptide followed by an N-terminal PFO domain that is fused to a C-terminal NADPH-cytochrome P450 reductase (CPR) domain. Two independent 5.8-kb full-size cDNAs for Euglena mitochondrial PNO were isolated; the gene was expressed in cultures supplied with 2% CO(2) in air and with 2% CO(2) in N(2). The apicomplexan Cryptosporidium parvum was also shown to encode and express the same PFO-CPR fusion, except that, unlike E. gracilis, no mitochondrial transit peptide for C. parvum PNO was found. Recombination-derived remnants of PNO are conserved in the genomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe as proteins involved in sulfite reduction. Notably, Trypanosoma brucei was found to encode homologs of both PFO and all four PDH subunits. Gene organization and phylogeny revealed that eukaryotic nuclear genes for mitochondrial, hydrogenosomal, and cytosolic PFO trace to a single eubacterial acquisition. These findings suggest a common ancestry of PFO in amitochondriate protists with Euglena mitochondrial PNO and Cryptosporidium PNO. They are also consistent with the view that eukaryotic PFO domains are biochemical relics inherited from a facultatively anaerobic, eubacterial ancestor of mitochondria and hydrogenosomes.

Multiple secondary origins of the anaerobic lifestyle in eukaryotes

“…Posterior probabilities for some groups are shown, with a value of 1.0 representing maximum support. The strongly supported groups have also been recovered in previously published analyses using different methods, including maximum-likelihood, and different support measures including bootstrapping (Horner et al 1999;Rotte et al 2001). examined (Katinka et al 2001;LaGier et al 2003; but only the Trichomonas hydrogenosome (Sutak et al 2004) and Giardia mitosome (Tovar et al 2003) have actually been demonstrated to contain the pathway. Entamoeba is so far unique among eukaryotes in that it has lost the mitochondrial homologues of IscS and IscU (Loftus et al 2005), and instead it possesses genes for the homologous proteins NifS and NifU, acquired through horizontal gene transfer from a bacterium related to Campylobacter (Ali et al 2004;van der Giezen et al 2004).…”

Mitochondrial trans-2-Enoyl-CoA Reductase of Wax Ester Fermentation from Euglena gracilis Defines a New Family of Enzymes Involved in Lipid Synthesis

“…The plasmid pEgTER1 contains an ORF of 539 aa with a calculated molecular mass of 59 kDa, substantially larger than the molecular mass of purified TER (44 kDa). Euglena mitochondrial targeting sequences do not belong to the learning set employed by targeting prediction programs such as MitoprotII (34) and iP-SORT (35) and only a few transit peptides of Euglena mitochondrial proteins are known, which are about 30 -40-aa long (8,36). Thus, the exact role of the 135 N-terminal aa in the ORF of pEgTER1 is unclear as is the identity of the start codon used in vivo.…”

“…The wax ester fatty acids are synthesized from acetyl-CoA stemming from pyruvate via an unusual oxygensensitive enzyme, pyruvate:NADP ϩ oxidoreductase (6,7), the core catalytic component of which is pyruvate:ferredoxin oxidoreductase, a typical enzyme of hydrogenosomes (8). Pyruvate:NADP ϩ oxidoreductase exists in the mitochondrion alongside a classical mitochondrial pyruvate dehydrogenase complex with mRNA expression patterns converse to that of pyruvate: NADP ϩ oxidoreductase in response to hypoxia (9).…”

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

“…Medium and Culture Conditions-E. gracilis strain Z (SAG 1224 -5/25 collection of algae Göttingen) for isolation of mitochondria and subsequent analysis by two-dimensional PAGE was cultured as described previously (6). Euglena cultures for determination of UQ 9 and RQ 9 were grown in a BIOSTAT B 10-liter fermenter (Braun Biotech) at a culturing volume of 7 liters with light continuously at 5000 lux, constant temperature at 28°C, and stirring at 200 rpm.…”

“…unusual, oxygen-sensitive enzyme, pyruvate:NADPϩ oxidoreductase (6,17). The resulting acetyl-CoA enters a modified citric acid cycle, which entails a shunt via succinate semialdehyde as in the ␣-proteobacterium Bradyrhizobium (18), circumventing the step catalyzed by ␣-ketoglutarate dehydrogenase.…”