Biosynthesis of Lipoic Acid in Arabidopsis: Cloning and Characterization of the cDNA for Lipoic Acid Synthase

Abstract

Lipoic acid is a coenzyme that is essential for the activity of enzyme complexes such as those of pyruvate dehydrogenase and glycine decarboxylase. We report here the isolation and characterization of LIP1 cDNA for lipoic acid synthase of Arabidopsis. The Arabidopsis LIP1 cDNA was isolated using an expressed sequence tag homologous to the lipoic acid synthase of Escherichia coli. This cDNA was shown to code for Arabidopsis lipoic acid synthase by its ability to complement a lipA mutant of E. coli defective in lipoic acid synthase. DNA-sequence analysis of the LIP1 cDNA revealed an open reading frame predicting a protein of 374 amino acids. Comparisons of the deduced amino acid sequence with those of E. coli and yeast lipoic acid synthase homologs showed a high degree of sequence similarity and the presence of a leader sequence presumably required for import into the mitochondria. Southernhybridization analysis suggested that LIP1 is a single-copy gene in Arabidopsis. Western analysis with an antibody against lipoic acid synthase demonstrated that this enzyme is located in the mitochondrial compartment in Arabidopsis cells as a 43-kD polypeptide.Lipoic acid (6,8-thioctic acid) is a sulfur-containing coenzyme that is required for the activity of enzyme complexes involved in the oxidative decarboxylation of ␣-ketoacids (Reed and Hackert, 1990;Perham, 1991;Mattevi et al., 1992) and in the Gly-cleavage system (Fujiwara et al., 1990;Kim and Oliver, 1990;Macherel et al., 1990). There are five lipoyl proteins: the dihydrolipoamide acyltransferase subunits of pyruvate, ␣-ketoglutarate, branched-chain ␣-ketoacid dehydrogenase complexes, protein X of the pyruvate dehydrogenase complex, and the H-protein of the Gly-cleavage system. Lipoic acid is covalently bound to these proteins via an amide linkage to the ⑀-amino group of specific Lys residues (Reed and Hackert, 1966). The lipoylLys arm functions as a carrier of reaction intermediates, and reducing equivalents interact with the active sites of the components of the complexes (Yeaman, 1989; Douce et al., 1994).Despite the importance of the lipoyl-prosthetic group in the functioning of several enzyme complexes, the biosynthesis of lipoic acid is not well understood in any organism. Molecular genetic studies (Vanden Boom et al., 1991;Reed and Cronan, 1993;Morris et al., 1994Morris et al., , 1995 of Escherichia coli have identified three genes, lipA, lipB, and lplA that are involved in the biosynthesis and transfer of lipoic acid. lipA encodes a lipoic acid synthase that is required for an insertion of the first sulfur atom into the octanoate backbone (Reed and Cronan, 1993). lplA and lipB encode lipoate ligases that are involved in the transfer of lipoic acid to cognate proteins (Morris et al., 1994(Morris et al., , 1995. LplA and LipB proteins primarily function in the utilization of lipoic acid exogenously added to the growth medium and endogenously synthesized lipoic acid in E. coli cells, respectively (Morris et al., 1994(Morris et al., , 1995. LplA protein is involved...

Discovery and Characterization of the 3-Hydroxyacyl-ACP Dehydratase Component of the Plant Mitochondrial Fatty Acid Synthase System

“…It is interesting that the mtFAS system appears to terminate the fatty acid elongation cycle via acyl transferase mechanisms, which is distinct from the ptFAS system that terminates the elongation cycle via a hydrolytic mechanism catalyzed by acyl-ACP thioesterases (Jing et al, 2011). Specifically, mtFAS uses the acyl-transferases AtLpxA, AtLpxD1, and AtLpxD2 (Li et al, 2011) for the assembly of lipid A-like molecules and a combination of lipoyl transferase (Wada et al, 2001) and lipoic acid synthase (Yasuno and Wada, 1998) to generate lipoic acid.…”

“…The mtFAS system appears to use free malonic acid as the substrate (Guan and Nikolau, 2016) to primarily generate octanoyl-acyl carrier protein (ACP), which is the required precursor for the biosynthesis of lipoic acid (Yasuno and Wada, 1998;Gueguen et al, 2000;Wada et al, 2001). Lipoic acid is the cofactor that is essential for pyruvate dehydrogenase (PDH), a-ketoglutarate dehydrogenase (KGDH), branchedchain a-ketoacid dehydrogenase, and the Gly decarboxylase complex (GDC; Taylor et al, 2004).…”

Mitochondrial Protein Lipoylation Does Not Exclusively Depend on the mtKAS Pathway of de Novo Fatty Acid Synthesis in Arabidopsis

“…Alternatively, and possibly more likely, octanoyl chains could be supplied from the b-oxidation of longer-chain fatty acids in peroxisomes (Graham and Eastmond, 2002;Rylott et al, 2006). Lipoate synthase has been identified in plant mitochondria (Yasuno and Wada, 1998); however, such a scavenging pathway would also require lipoyl-protein ligase (LPLA). While such enzymes were unknown in plants until very recently, a bacterialtype bifunctional LPLA of yet unknown subcellular location has been identified in rice (Oryza sativa; Kang et al, 2007).…”

Identification and Molecular Characterization of the β-Ketoacyl-[Acyl Carrier Protein] Synthase Component of the Arabidopsis Mitochondrial Fatty Acid Synthase

“…Total RNAs used for RT-PCR were extracted from leaves, roots, and flowers of 4-week-old A. thaliana with an RNA extraction kit (RNeasy® Plant Mini Kit; Qiagen). The primer set used for amplifying and cloning of the mtKAS-⌬20 sequence was used for RT-PCR as described before (39). As a control the expression of the gene for ␣-tubulin of A. thaliana (40) was also checked with the same RNA preparations using the primer set 5Ј-CTACTGAGAGAAG-ATGCGAG-3Ј and 5Ј-CAACATCTCCTCGGTACATC-3Ј.…”

“…The obtained plasmid was used to transform E. coli BL21(DE3) (41). Expression of mtKAS-⌬20 in BL21(DE3), purification of mtKAS-⌬20 protein, and production of a polyclonal antibody against this protein were performed using a previously described procedure (39).…”