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

• Published in: The Journal of biological chemistry Vol. 281; no. 47; pp. 35894 - 35903
• Main Authors: Goepfert, Simon, Hiltunen, J. Kalervo, Poirier, Yves
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 24.11.2006; American Society for Biochemistry and Molecular Biology
• Subjects: Allium cepa; Amino Acid Sequence; Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis Proteins - chemistry; Arabidopsis Proteins - genetics; Arabidopsis thaliana; Carbon - chemistry; Catalysis; Enoyl-CoA Hydratase - chemistry; Enoyl-CoA Hydratase - genetics; Fatty Acids - chemistry; Fatty Acids, Unsaturated - chemistry; Molecular Sequence Data; Onions; Oxygen - chemistry; Peroxisomes - chemistry; Peroxisomes - enzymology; Plant Proteins - chemistry; Recombinant Fusion Proteins - chemistry; Saccharomyces cerevisiae; Saccharomyces cerevisiae - metabolism; Sequence Homology, Amino Acid
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M606383200

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 β-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 β-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 β-oxidation cycle.