The origin of pyruvate:NADP+ oxidoreductase in mitochondria of Euglena gracilis (2000)

Euglena gracilis, a protist having chloroplasts, has unique mitochondria, in which the common pyruvate and 2-oxoglutrate dehydrogenase multienzyme complexes are absent [1]. In the mitochondria, 2-oxoglutrate is converted to succinate through succinate semialdehyde by the sequential reactions of 2-oxoglutrate decarboxylase and succinate semialdehyde dehydrogenase. In contrast, pyruvate is oxidized in a CoA-dependent reaction to form acetyl-CoA and CO2 by the action of a single enzyme, pyruvate:NADP+ oxidoreductase (PNO) (EC 1.2.1.51), with NADP+ (but not NAD+) as an electron acceptor. It is suggested that these three enzymes participate in the respiratory metabolism of this organism. PNO, in contrast to the other two enzymes, has not yet been found in any other organisms. These unique characteristics of Euglena provide insight regarding the origin of mitochondria. In this communication, we propose that PNO was evolved from pyruvate:ferredoxin oxidoreductase (PFO) by linking with a flavoprotein with both FMN and FAD at the C-terminal side by gene fusion. To address the origin of PNO, we cloned cDNA encoding this enzyme and analyzed it. The PNO cDNA (5861 bp) had an open reading frame encoding a polypeptide with 1803 amino acid residues. The N-terminal amino acid in the mature form of this enzyme (Thr [1]) was found at the position of 38 in the deduced amino acid sequence of the cDNA. It is thus indicated that the mature form of PNO, a homodimeric protein, consists of 1766 amino acid residues per subunit (calculated subunit molecular weight: 195 536), and the precursor form of this enzyme has a signal sequence with 37 amino acid residues for targeting to mitochondria. We have reported that limited proteolysis of PNO with trypsin gives two functionally active fragments [1]. One of the two fragments, derived from the N-terminal side of this enzyme, retains a homodimeric structure, and is still capable of catalyzing the CoA-dependent oxidative decarboxylation of pyruvate when an artificial electron acceptor, such as methyl viologen, is substituted for NADP+. Another fragment (the C-terminal fragment) is a monomeric peptide and retains an electron-transfer activity from NADPH to methyl viologen or other artificial electron acceptors. In the deduced amino acid sequence of the PNO cDNA, the N-terminal amino acid of the C-terminal fragment (Ala [1]) was found at the position of 1240. The amino acid sequence of the N-terminal fragment of PNO (amino acid position in the deduced amino acid sequence of the cDNA: 38–1239) closely resembled the sequences of homodimeric PFOs found in amitochondriate eukaryote Entamoeba histolytica (53% positional identity) and Trichomonas vaginalis (44%), and in eubacterium Rhodospirillum rubrum (44%), Klebsiella pneumoniae (49%) and Desulfovibrio africanus (53%). Similar to these homodimeric PFORs [2], this result shows that the N-terminal part of PNOR likely contains in each subunit one TBP, two ferredoxin-type [4Fe–4S] clusters, and one [4Fe–4S] cluster bound to an atypical cysteine-containing sequence motif. In contrast, the C-terminal fragment of PNO (amino acid position: 1240–1803) showed structural similarity to mammalian NADPH-cytochrome P450 reductase (positional identity to rat enzyme: 28%) and to bacterium NADPH-sulfite reductase α subunit (positional identity to Salmonella typhimurium enzyme: 21%). This finding suggests that the C-terminal part of PNOR, as well as NADPH-cytochrome P450 reductase and NADPH-sulfite reductase α subunit [3], has FMN and FAD (each one per subunit). It is thus suggested that each subunit (about 195 kDa) of PNO, a homodimeric protein, consists of two functional components, Fe–S domain (about 130 kDa) and flavodomain (about 65 kDa); the Fe–S domain locates at the N-terminal side and is linked with the flavodomain by a trypsin-sensitive hinge region (Fig. 1). The reaction mechanism of this enzyme is thought as follows: On the Fe–S domain, pyruvate is oxidized in a CoA-dependent reaction to form acetyl-CoA and CO2 by the cooperation of TBP and [4Fe–4S] clusters. Two electrons formed by the pyruvate oxidation in the Fe–S domain are once transferred to the flavodomain with FMN and FAD as redox centers, then accepted by NADP+. In archaea, most anaerobic eubacteria and amitochondriate eukaryotes, PFO acts for the oxidative decarboxylation of pyruvate to form acetyl-CoA, and the two electrons formed are accepted by ferredoxin with a more negative redox potential than that of NAD+ [2]. This is in contrast to most respiratory organisms having the pyruvate dehydrogenase multienzyme complex. On the basis of subunit structure, PFOs are divided into homodimeric, heterodimeric and heterotetrameric types. These three types of PFO are phylogenetically close and their evolution is explained by the rearrangement and eventual fusion of four ancestral genes. Amitochondriate eukaryotes have homodimeric PFO, and this enzyme plays a central role in anaerobic energy metabolism [4, 5]. In particular, in amitochondriate eukaryotes having hydrogenosomes (the double-membrane-bound, H2- and ATP-producing organelles), such as Trichomonas, PFO is located in the organelles and participates, together with hydrogenase, in the formation of acetate and H2 from pyruvate, yielding ATP by substrate-level phosphorylation. On the basis of this evidence, we propose that PNO was evolved by linking with a homodimeric PFO and a flavoenzyme (which is the same origin as NADPH-cytochrome P450 reductase and NADPH-sulfite reductase α subunit) by gene fusion. In other words, the ferredoxin-type enzyme was converted to the NADP+-dependent one by linking covalently with a flavoprotein with both FMN and FAD at the C-terminal side. In addition, it is thought that this conversion was necessary to make it possible to participate in the respiratory metabolism in the mitochondria of E. gracilis. In the hydrogen hypothesis for the first eukaryote, mitochondria and hydrogenosomes are proposed to originate from a common ancestor [5]. Thus, the occurrence of PNO in Euglena mitochondria is interesting in understanding the relationship between mitochondria and hydrogenosomes.