Analysis of the Alternative Pathways for the β-Oxidation of Unsaturated Fatty Acids Using Transgenic Plants Synthesizing Polyhydroxyalkanoates in Peroxisomes

Allenbach, Laure; Poirier, Yves

doi:10.1104/pp.124.3.1159

Cited by 34 publications

(13 citation statements)

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“…The contribution of AtECH2 to the degradation of such unsaturated fatty acids was assessed by the use of PHA analysis. PHA biosynthesis in the peroxisome of plants and yeasts has previously been shown to be a useful tool to study the in vivo carbon flux through the ␤-oxidation cycle (28,32,34,40,41).…”

Section: Discussionmentioning

confidence: 99%

“…6 and 7) (40). Although the presence of the monomer 3-hydroxy,4Z-undecenoic acid in PHA derived from the degradation of 10Z-heptadecenoic acid would have provided direct evidence for the hydratase-epimerase pathway, 3-hydroxyacyl-CoA monomers having an unsaturated bond between the third and fourth carbon are not substrates to the PHA synthase (34,40,41). However, the increase in the proportion of the H9:0 monomer relative to H7:0 and H11:0 in PHA derived from the degradation of 10Z-heptadecenoic acid compared with PHA derived from 10E-heptadecenoic acid (Figs.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, 3-hydroxyacyl-CoA containing a double bond on carbon 4 has been reported to be a poor substrate for the PHA synthase (shown in parentheses in Fig. 6) and are typically not found in PHA (34,40,41).…”

Section: Modulation Of the Carbon Flux Through ␤-Oxidation In Transgementioning

confidence: 99%

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Identification and Functional Characterization of a Monofunctional Peroxisomal Enoyl-CoA Hydratase 2 That Participates in the Degradation of Even cis-Unsaturated Fatty Acids in Arabidopsis thaliana

Goepfert

Hiltunen

Poirier

2006

Journal of Biological Chemistry

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A gene, named AtECH2, has been identified in Arabidopsis thaliana to encode a monofunctional peroxisomal enoyl-CoA hydratase 2. Homologues of AtECH2 are present in several angiosperms belonging to the Monocotyledon and Dicotyledon classes, as well as in a gymnosperm. In vitro enzyme assays demonstrated that AtECH2 catalyzed the reversible conversion of 2E-enoyl-CoA to 3R-hydroxyacyl-CoA. AtECH2 was also demonstrated to have enoyl-CoA hydratase 2 activity in an in vivo assay relying on the synthesis of polyhydroxyalkanoate from the polymerization of 3R-hydroxyacyl-CoA in the peroxisomes of Saccharomyces cerevisiae. AtECH2 contained a peroxisome targeting signal at the C-terminal end, was addressed to the peroxisome in S. cerevisiae, and a fusion protein between AtECH2 and a fluorescent protein was targeted to peroxisomes in onion cells. AtECH2 gene expression was strongest in tissues with high ␤-oxidation activity, such as germinating seedlings and senescing leaves. The contribution of AtECH2 to the degradation of unsaturated fatty acids was assessed by analyzing the carbon flux through the ␤-oxidation cycle in plants that synthesize peroxisomal polyhydroxyalkanoate and that were over-or underexpressing the AtECH2 gene. These studies revealed that AtECH2 participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2E-enoyl-CoA for further degradation through the core ␤-oxidation cycle.The peroxisome is the site of numerous important biochemical reactions in plants, including photorespiration, the ␤-oxidation cycle, and the glyoxylate cycle. Although several enzymes involved in these pathways have been identified, analysis of the plant proteome for proteins possessing putative peroxisome targeting sequences have identified numerous candidate peroxisomal proteins for which no functions have been assigned (1). Furthermore, prediction of peroxisomal proteins can be made difficult by the absence of recognizable signal peptide, particularly for peroxisomal membrane protein. Thus, our present knowledge of the complexity of the biochemical pathways present in the peroxisome is fragmented.The peroxisomal ␤-oxidation cycle is of primary importance during seedling establishment following germination, because it is responsible for the breakdown of fatty acids into acetylCoA, which is subsequently converted to glucose via the glyoxylate cycle and gluconeogenesis (2). Although fatty acid ␤-oxidation is very active during germination in oleaginous seed and during senescence, this cycle is also present in mature photosynthetic tissues, such as leaves, as well as in developing seeds (3).Degradation of saturated fatty acids in the peroxisome occurs via four enzyme activities located on three proteins that form the core ␤-oxidation pathway. The first step is mediated by an acyl-CoA oxidase, converting acyl-CoA to 2E-enoyl-CoA. This is followed by the hydration of the 2E-enoyl-CoA to 3-hydroxyacyl-CoA by an...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Identification and Functional Characterization of a Monofunctional Peroxisomal Enoyl-CoA Hydratase 2 That Participates in the Degradation of Even cis-Unsaturated Fatty Acids in Arabidopsis thaliana

Goepfert

Hiltunen

Poirier

2006

Journal of Biological Chemistry

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“…The same was observed in mcl-PHAs accumulated in natural or recombinant prokaryotes (Table 1). In contrast, when b-oxidation intermediates were directed into mcl-PHA biosynthesis in natural hosts (De Waard et al 1993), recombinant yeast (Poirier et al 2001 or recombinant A. thaliana (Mittendorf et al 1998(Mittendorf et al , 1999Allenbach and Poirier 2000), the monomer composition also included unsaturated monomers.…”

Section: Discussionmentioning

confidence: 99%

Expression of poly-3-(R)-hydroxyalkanoate (PHA) polymerase and acyl-CoA-transacylase in plastids of transgenic potato leads to the synthesis of a hydrophobic polymer, presumably medium-chain-length PHAs

Romano

Plas

Witholt³

et al. 2004

Planta

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Medium-chain-length poly-3-(R)-hydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The minimum gene-set for the accumulation of mcl-PHAs from de novo fatty acid biosynthesis has been identified in prokaryotes [B. Rehm et al. (1998) J. Biol Chem 273:24044-24051] as consisting of the Pha-C1 polymerase and the ACP-CoA-transacylase. In this paper, the synthesis of mcl-PHAs has been attempted in transgenic potato (Solanum tuberosum L.) using the same set of genes that were introduced into potato by particle bombardment. Polymer contents of transgenic lines were analysed by gas chromatography and by a new simple method employing a size-exclusion filter column. The expression of the Pha-C1 polymerase and the ACP-CoA-transacylase in the plastids of transgenic potato led to the synthesis of a hydrophobic polymer composed of mcl-hydroxy-fatty acids with carbon chain lengths ranging from C-6 to C-12 in leaves of the selected transgenic lines. We strongly suggest that the polymer observed consists of mcl-PHAs and that this report establishes for the first time a possible route for the production of mcl-PHAs from de novo fatty acid biosynthesis in plants.

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“…From these initial studies, peroxisomal PHA was subsequently used to reveal the existence of a futile cycle of fatty acids in transgenic plants expressing a medium-chain fatty acyl-acyl carrier protein thioesterase in leaves or developing seeds (15,16) as well as in developing A. thaliana embryos deficient in the synthesis of triacylglycerides (16). The PHA monomer composition has also been used to analyze the pathway of degradation of unsaturated fatty acids in plants (17).…”

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Futile Cycling of Intermediates of Fatty Acid Biosynthesis toward Peroxisomal β-Oxidation in Saccharomyces cerevisiae

Marchesini

Poirier

2003

Journal of Biological Chemistry

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The flux of fatty acids toward ␤-oxidation was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate synthesis in the peroxisome from the polymerization, by a bacterial polyhydroxyalkanoate synthase, of the ␤-oxidation intermediates 3-hydroxyacyl-CoAs. Synthesis of polyhydroxyalkanoate was dependent on the ␤-oxidation enzymes acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase multifunctional protein, which are involved in generating 3-hydroxyacyl-CoAs, and on the peroxin PEX5, which is involved in the import of proteins into the peroxisome. In wild type cells grown in media containing fatty acids, the polyhydroxyalkanoate monomer composition was largely influenced by the nature of the external fatty acid, such that even-chain monomers are generated from oleic acid and odd-chain monomers are generated from heptadecenoic acid. In contrast, polyhydroxyalkanoate containing predominantly 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate was synthesized in a mutant deficient in the peroxisomal 3-ketothiolase (fox3⌬0) growing either on oleic acid or heptadecenoic acid as well as in wild type and fox3⌬0 mutants grown on glucose or raffinose, indicating that 3-hydroxyacyl-CoAs used for polyhydroxyalkanoate synthesis were generated from the degradation of intracellular short-and medium-chain fatty acids by the ␤-oxidation cycle. Inhibition of fatty acid biosynthesis with cerulenin blocked the synthesis of polyhydroxyalkanoate from intracellular fatty acids but still enabled the use of extracellular fatty acids for polymer production. Mutants affected in the synthesis of lipoic acid showed normal polyhydroxyalkanoate synthesis capacity. Together, these results uncovered the existence of a substantial futile cycle whereby short-and mediumchain intermediates of the cytoplasmic fatty acid biosynthetic pathway are directed toward the peroxisomal ␤-oxidation pathway.

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Analysis of the Alternative Pathways for the β-Oxidation of Unsaturated Fatty Acids Using Transgenic Plants Synthesizing Polyhydroxyalkanoates in Peroxisomes

Cited by 34 publications

References 34 publications

Identification and Functional Characterization of a Monofunctional Peroxisomal Enoyl-CoA Hydratase 2 That Participates in the Degradation of Even cis-Unsaturated Fatty Acids in Arabidopsis thaliana

Identification and Functional Characterization of a Monofunctional Peroxisomal Enoyl-CoA Hydratase 2 That Participates in the Degradation of Even cis-Unsaturated Fatty Acids in Arabidopsis thaliana

Expression of poly-3-(R)-hydroxyalkanoate (PHA) polymerase and acyl-CoA-transacylase in plastids of transgenic potato leads to the synthesis of a hydrophobic polymer, presumably medium-chain-length PHAs

Futile Cycling of Intermediates of Fatty Acid Biosynthesis toward Peroxisomal β-Oxidation in Saccharomyces cerevisiae

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