Fatty acid and lipoic acid biosynthesis were investigated in plant mitochondria. Although the mitochondria lack acetyl-CoA carboxylase, our experiments reveal that they contain the enzymatic equipment necessary to transform malonate into the two main building units for fatty acid synthesis: malonyl-and acetyl-acyl carrier protein (ACP). We demonstrated, by a new method based on a complementary use of high performance liquid chromatography and mass spectrometry, that the soluble mitochondrial fatty-acid synthase produces mainly three predominant acyl-ACPs as follows: octanoyl(C8)-, hexadecanoyl(C16)-, and octadecanoyl (C18)-ACP. Octanoate production is of primary interest since it has been postulated long ago to be a precursor of lipoic acid. By using a recombinant H apoprotein mutant as a potential acceptor for newly synthesized lipoic acid, we were able to detect limited amounts of lipoylated H protein in the presence of malonate, several sulfur donors, and cofactors. Finally, we present a scheme outlining the new biochemical pathway of fatty acid and lipoic acid synthesis in plant mitochondria.Lipoic acid (6,8-thioctic acid or 1,2-dithiolane-3-pentanoic acid) is a sulfur-containing cofactor involved in several multienzyme complexes such as pyruvate dehydrogenase, ␣-ketoglutarate dehydrogenase, branched-chain keto acid dehydrogenase, and glycine decarboxylase complex. The carboxyl group of lipoic acid is attached to the dihydrolipoamide acyltransferase subunits (E 2 ) of the keto acid dehydrogenase complexes and to the H protein of the glycine decarboxylase complex, by an amide linkage to the ⑀-amino group of a specific lysine (1-4).Recent studies have highlighted the potential of free lipoic acid and dihydrolipoic acid as powerful metabolic antioxidants that are able to scavenge the reactive oxygen species, to recycle other antioxidants (vitamin C, glutathione, and vitamin E), and even to intervene in redox regulation of gene transcription (5, 6). Consequently, lipoic acid is now increasingly used as a therapeutic agent in pathologies associated with oxidative stress (for a review see Packer et al. (5)).In prokaryotic cells, Parry (7) and White (8) showed by labeling experiments that octanoic acid was a direct precursor of lipoic acid, 6-thiooctanoate and 8-thiooctanoate being possible intermediates in lipoic acid biosynthesis (9 -11). Mutant strains of Escherichia coli defective in lipoic acid biosynthesis have allowed the isolation of several genes involved in lipoic acid biosynthesis (12)(13)(14). The characterization of the lip locus revealed that it contained the lipA gene encoding for a 36-kDa protein (14 -16). Despite the fact that LipA activity has never been measured in vitro, the protein is expected to be related to a lipoate synthase. Sequence similarity to biotin synthase strongly suggests that lipA encodes a sulfur insertion enzyme analogous to biotin synthase and, consequently, that the sulfur insertion mechanisms of the two systems could be related (15,16). Moreover, biotin synthase is known to ...

The mitochondrial glycine decarboxylase complex (GDC) consists of four component enzymes (P, H, T, and L proteins) involved in the breakdown of glycine. In order to investigate structural interactions involved in the stabilization of the methylamine-loaded H protein (a transient species in the GDC reaction), we designed several mutants of H apoprotein.Structural analysis of the wild-type and mutants of H apoprotein emphasized the necessity to carefully assess, by biophysical techniques, the correct folding of mutated proteins prior to investigate their biochemical properties. The correctly folded wild-type and mutants of H apoprotein were in vitro lipoylated and then characterized in the context of GDC reaction by studying the reconstituted complex and partial reactions. We showed that Val 62 and Ala 64 , surrounding the lipoyl-lysine, play an important role in the molecular events that govern the reaction between P and H protein but do not intervene in the recognition of the binding site of lipoic acid by lipoyl ligase. The biochemical results obtained with the HE14A mutant of H protein pointed out the major role of the Glu 14 amino acid residue in the GDC catalysis and highlighted the importance of the ionic and hydrogen bounds in the hydrophobic cleft of H protein for the stabilization of the methylamine-loaded lipoyl arm.