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

Abstract

Under anaerobiosis, Euglena gracilis mitochondria perform a malonyl-CoA independent synthesis of fatty acids leading to accumulation of wax esters, which serve as the sink for electrons stemming from glycolytic ATP synthesis and pyruvate oxidation. An important enzyme of this unusual pathway is trans-2-enoyl-CoA reductase (EC 1.3.1.44), which catalyzes reduction of enoyl-CoA to acyl-CoA. Trans-2-enoyl-CoA reductase from Euglena was purified 1700-fold to electrophoretic homogeneity and was active with NADH and NADPH as the electron donor. The active enzyme is a monomer with molecular mass of 44 kDa. The amino acid sequence of tryptic peptides determined by electrospray ionization mass spectrometry were used to clone the corresponding cDNA, which encoded a polypeptide that, when expressed in Escherichia coli and purified by affinity chromatography, possessed trans-2-enoyl-CoA reductase activity close to that of the enzyme purified from Euglena. Trans-2-enoyl-CoA reductase activity is present in mitochondria and the mRNA is expressed under aerobic and anaerobic conditions. Using NADH, the recombinant enzyme accepted crotonyl-CoA (k m ‫؍‬ 68 M) and trans-2-hexenoyl-CoA (k m ‫؍‬ 91 M). In the crotonyl-CoA-dependent reaction, both NADH (k m ‫؍‬ 109 M) or NADPH (k m ‫؍‬ 119 M) were accepted, with 2-3-fold higher specific activities for NADH relative to NADPH. Trans-2-enoyl-CoA reductase homologues were not found among other eukaryotes, but are present as hypothetical reading frames of unknown function in sequenced genomes of many proteobacteria and a few Gram-positive eubacteria, where they occasionally occur next to genes involved in fatty acid and polyketide biosynthesis. Trans-2-enoyl-CoA reductase assigns a biochemical activity, NAD(P)H-dependent acyl-CoA synthesis from enoylCoA, to one member of this gene family of previously unknown function.

Biochemical characterization of a mitochondrial-like organelle from Blastocystis sp. subtype 7

“…This finding is consistent with the proposed common origin of hydrogenosomes and mitochondria (Martin, 1999). The absence of detectable succinate dehydrogenase activity suggests that the primary function of the partial Krebs cycle in Blastocystis is to provide precursors for the synthesis of amino acids or acetyl-CoA for fatty acid biosynthesis as occurs in E. gracilis (Hoffmeister et al, 2005). The presence of a partial Krebs cycle has been reported in the mitochondria from procyclic Trypanosoma brucei (Van Hellemond et al, 2005;van Weelden et al, 2005), which is proposed to function to export acetyl-CoA to the cytoplasm for fatty acid biosynthesis.…”

Euglena gracilis Ribonucleotide Reductase

“…Hence, the presence of a clearly homologous RNR gene in the two lineages could suggest that both a class I and II RNR was present in the common ancestor of all eukaryotes (vertical descent) followed by differential loss in several independent lineages. Euglena sustains prolonged anaerobiosis with the help of a highly adapted wax ester fermentation (40,41). Although the slime mold Dictyostelium is not a strict anaerobe, its relatives Entamoeba and Mastigamoeba are (42).…”

Retro-biosynthetic screening of a modular pathway design achieves selective route for microbial synthesis of 4-methyl-pentanol

“…To the best of our knowledge, 3-hydroxy-acylCoA dehydratases and trans-enoyl reductases with activity on the subsequent branched intermediates have not been identified. From enzymes documented to have activity on straight medium-chain CoA substrates, 4 phaJ and 6 ter homologues were selected for further screening 48,49 (Supplementary Table 1). An assay was developed to screen for enzymes with the desired activity by isolating modules 2 and 3 of our pathway in vivo with different combinations of dehydratases and reductases.…”