Occurrence of 5'-Deoxyadenosylcobalamin and Its Physiological Function as the Coenzyme of Methylmalonyl-CoA Mutase in a Marine Eukaryotic Microorganism, Schizochytrium limacinum SR21

Miyamoto, Emi; Tanioka, Yuri; Yukino, Tsugiyo; Hayashi, Masahiro; Watanabe, Fumio; Nakano, Yoshihisa

doi:10.3177/jnsv.53.471

Cited by 5 publications

(3 citation statements)

References 18 publications

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“…PLAT1 contained two transmembrane domains and four AGPAT motifs found in LPCATs [22], [25], [27]. Three EF hand motifs and a KK motif, which could serve as a calcium-binding motif and ER-retaining motif, respectively, were located at the C terminus (Figure 3).…”

Section: Resultsmentioning

confidence: 99%

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Novel Lysophospholipid Acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b Responsible for Generation of Palmitate-Docosahexaenoate-Phosphatidylcholine and Phosphatidylethanolamine

et al. 2014

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N-3 polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA, 22:6n-3), have been reported to play roles in preventing cardiovascular diseases. The major source of DHA is fish oils but a recent increase in the global demand of DHA and decrease in fish stocks require a substitute. Thraustochytrids, unicellular marine protists belonging to the Chromista kingdom, can synthesize large amounts of DHA, and, thus, are expected to be an alternative to fish oils. DHA is found in the acyl chain(s) of phospholipids as well as triacylglycerols in thraustochytrids; however, how thraustochytrids incorporate DHA into phospholipids remains unknown. We report here a novel lysophospholipid acyltransferase (PLAT1), which is responsible for the generation of DHA-containing phosphatidylcholine and phosphatidylethanolamine in thraustochytrids. The PLAT1 gene, which was isolated from the genomic DNA of Aurantiochytrium limacinum F26-b, was expressed in Saccharomyces cerevisiae, and the FLAG-tagged recombinant enzyme was characterized after purification with anti-FLAG affinity gel. PLAT1 shows wide specificity for donor substrates as well as acceptor substrates in vitro, i.e, the enzyme can adopt lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylserine and lysophosphatidylinositol as acceptor substrates, and 15:0/16:0-CoA and DHA-CoA as donor substrates. In contrast to the in vitro experiment, only lysophosphatidylcholine acyltransferase and lysophosphatidylethanolamine acyltransferase activities were decreased in plat1-knockout mutants, resulting in a decrease of 16:0-DHA-phosphatidylcholine (PC) [PC(38∶6)] and 16:0-DHA-phosphatidylethanolamine (PE) [PE(38∶6)], which are two major DHA-containing phospholipids in A. limacinum F26-b. However, the amounts of other phospholipid species including DHA-DHA-PC [PC(44∶12)] and DHA-DHA-PE [PE(44∶12)] were almost the same in plat-knockout mutants and the wild-type. These results indicate that PLAT1 is the enzyme responsible for the generation of 16:0-DHA-PC and 16:0-DHA-PE in the thraustochytrid.

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Section: Resultsmentioning

confidence: 99%

“…However, most of 15∶0 was replaced by palmitic acid (16∶0) with the addition of vitamin B12. The odd-chain fatty acid (15∶0) could also be generated from the fatty acid synthase (FAS) pathway using propionyl-CoA as a primer instead of malonyl-CoA in the absence of vitamin B12 [24], [25].…”

Section: Discussionmentioning

confidence: 99%

Novel Lysophospholipid Acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b Responsible for Generation of Palmitate-Docosahexaenoate-Phosphatidylcholine and Phosphatidylethanolamine

et al. 2014

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“…The apparent molecular mass of Euglena MCM was found to be 149.0 kDa and 75.0 kDa using Superdex 200 gel filtration and SDS-PAGE, respectively, indicating that the Euglena enzyme is composed of two identical subunits (Miyamoto et al 2010). The enzyme contained one mole of prosthetic AdoB12 per mole of the enzyme subunit similar to AdoB12 content of the human liver (Fenton et al 1982), intestinal worm (Han et al 1984), and other algal (Miyamoto et al 2004(Miyamoto et al , 2007 MCMs. Euglena MCM encodes a putative mitochondrial targeting signal consisting of nine amino acids.…”

Section: Vitamin B12mentioning

confidence: 93%

Biochemistry and Physiology of Vitamins in Euglena

Watanabe

Yoshimura

Shigeoka

2017

Advances in Experimental Medicine and Biology

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Euglena gracilis Z requires vitamins B1 and B12 for growth. It takes up and accumulates large amounts of these exogenous vitamins through energy-dependent active transport systems. Except for these essential vitamins, E. gracilis Z has the ability to synthesize all human vitamins. Euglena synthesizes high levels of antioxidant vitamins such as vitamins C and E, and, thus, are used as nutritional supplements for humans and domestic animals. Methods to effectively produce vitamins in Euglena have been investigated.Previous biochemical studies indicated that E. gracilis Z contains several vitamin-related novel synthetic enzymes and metabolic pathways which suggests that it is a highly suitable organism for elucidating the physiological functions of vitamins in comparative biochemistry and biological evolution. E. gracilis Z has an unusual biosynthetic pathway for vitamin C, a hybrid of the pathways found in animals and plants. This chapter presents up-to-date information on the biochemistry and physiological functions of vitamins in this organism.

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Characterization of methylmalonyl-CoA mutase involved in the propionate photoassimilation of Euglena gracilis Z

et al. 2010

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Significant accumulation of the methylmalonyl-CoA mutase apoenzyme was observed in the photosynthetic flagellate Euglena gracilis Z at the end of the logarithmic growth phase. The apoenzyme was converted to a holoenzyme by incubation for 4 h at 4 degrees C with 10 microM 5'-deoxyadenosylcobalamin, and then, the holoenzyme was purified to homogeneity and characterized. The apparent molecular mass of the enzyme was calculated to be 149.0 kDa +/- 5.0 kDa using Superdex 200 gel filtration. SDS-polyacrylamide gel electrophoresis of the purified enzyme yielded a single protein band with an apparent molecular mass of 75.0 kDa +/- 3.0 kDa, indicating that the Euglena enzyme is composed of two identical subunits. The purified enzyme contained one mole of prosthetic 5'-deoxyadenosylcobalamin per mole of the enzyme subunit. Moreover, we cloned the full-length cDNA of the Euglena enzyme. The cDNA clone contained an open reading frame encoding a protein of 717 amino acids with a calculated molecular mass of 78.3 kDa, preceded by a putative mitochondrial targeting signal consisting of nine amino acid residues. Furthermore, we studied some properties and physiological function of the Euglena enzyme.

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Occurrence of 5'-Deoxyadenosylcobalamin and Its Physiological Function as the Coenzyme of Methylmalonyl-CoA Mutase in a Marine Eukaryotic Microorganism, Schizochytrium limacinum SR21

Cited by 5 publications

References 18 publications

Novel Lysophospholipid Acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b Responsible for Generation of Palmitate-Docosahexaenoate-Phosphatidylcholine and Phosphatidylethanolamine

Novel Lysophospholipid Acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b Responsible for Generation of Palmitate-Docosahexaenoate-Phosphatidylcholine and Phosphatidylethanolamine

Biochemistry and Physiology of Vitamins in Euglena

Characterization of methylmalonyl-CoA mutase involved in the propionate photoassimilation of Euglena gracilis Z

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