Publications

• Purification and characterization of pyruvate:NADP+ oxidoreductase in Euglena gracilis.

  Inui H., Ono K., Miyatake K., Nakano Y., Kitaoka S.

  Pyruvate:NADP+ oxidoreductase was homogeneously purified from crude extract of Euglena gracilis. The Mr of the enzyme was estimated to be 309,000 by gel filtration. The enzyme migrated as a single protein band with Mr of 166,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggest ... >> More

  Pyruvate:NADP+ oxidoreductase was homogeneously purified from crude extract of Euglena gracilis. The Mr of the enzyme was estimated to be 309,000 by gel filtration. The enzyme migrated as a single protein band with Mr of 166,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme consists of two identical polypeptides. The absorption spectrum of the native enzyme exhibited maxima at 278, 380, and 430 nm, and a broad shoulder was observed around 480 nm; the maximum at 430 nm was eliminated by reduction of the enzyme with dithionite. Reduction of the enzyme with pyruvate and CoA and reoxidation with NADP+ were proved from changes of absorption spectra. The enzyme contained 2 molecules of FAD and 8 molecules of iron. It was also indicated that the enzyme was thiamine pyrophosphate-dependent. The enzyme was oxygen-sensitive, and the reaction was affected by the presence of oxygen. Pyruvate was the most active substrate, but the enzyme was slightly active for 2-oxobutyrate, 3-hydroxypyruvate, and oxalacetate, but not for glyoxylate and 2-oxoglutarate. The native electron acceptor was NADP+, whereas NAD+ was completely inactive. Methyl viologen, benzyl viologen, FAD, and FMN were utilized as artificial electron acceptors, whereas spinach and Clostridium ferredoxins were inactive. Pyruvate synthesis by reductive carboxylation of acetyl-CoA with NADPH as the electron donor occurred by the reverse reaction of the enzyme. The enzyme also catalyzed a pyruvate-CO2 exchange reaction and electron-transfer reaction from NADPH to other electron acceptors like methyl viologen. These results indicate that pyruvate:NADP+ oxidoreductase in E. gracilis is clearly distinct from either the pyruvate dehydrogenase multienzyme complex or pyruvate:ferredoxin oxidoreductase. << Less

  J. Biol. Chem. 262:9130-9135(1987) [PubMed] [EuropePMC]

• 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.

  Rotte C., Stejskal F., Zhu G., Keithly J.S., Martin W.

  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 ... >> More

  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. << Less

  Mol. Biol. Evol. 18:710-720(2001) [PubMed] [EuropePMC]

• Occurrence of oxygen-sensitive, NADP+-dependent pyruvate dehydrogenase in mitochondria of Euglena gracilis.

  Inui H., Miyatake K., Nakano Y., Kitaoka S.

  Pyruvate dehydrogenase found in mitochondria of Euglena gracilis was active on NADP+ but not NAD+, and FAD and methyl viologen also served as electron acceptors. For 2-oxoglutarate dehydrogenase both NAD+ and NADP+ were utilized and the ratio of its activity on NAD+ and NADP+ was about 1:5. The ac ... >> More

  Pyruvate dehydrogenase found in mitochondria of Euglena gracilis was active on NADP+ but not NAD+, and FAD and methyl viologen also served as electron acceptors. For 2-oxoglutarate dehydrogenase both NAD+ and NADP+ were utilized and the ratio of its activity on NAD+ and NADP+ was about 1:5. The activity of pyruvate dehydrogenase was inhibited by pyruvate in aerobiosis, while not in anaerobiosis. << Less

  J Biochem 96:931-934(1984) [PubMed] [EuropePMC]

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