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.
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 beta-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 beta-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 beta-oxidation cycle.
Goepfert
Simon
S
Département de Biologie Moléculaire Végétale, Biophore Building, Université de Lausanne, CH-1015, Switzerland.
Hiltunen
J Kalervo
JK
Poirier
Yves
Y
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics*; D002244:Carbon / Q000737:chemistry; D002384:Catalysis; D004746:Enoyl-CoA Hydratase / Q000737:chemistry* / Q000235:genetics; D005227:Fatty Acids / Q000737:chemistry; D005231:Fatty Acids, Unsaturated / Q000737:chemistry*; D008969:Molecular Sequence Data; D019697:Onions; D010100:Oxygen / Q000737:chemistry; D020675:Peroxisomes / Q000737:chemistry / Q000201:enzymology*; D010940:Plant Proteins / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D029681:Arabidopsis Proteins; D005227:Fatty Acids; D005231:Fatty Acids, Unsaturated; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D002244:Carbon; C515968:ECH2 protein, Arabidopsis; D004746:Enoyl-CoA Hydratase; D010100:Oxygen