Abstract
Lipoic acid (LA, 6,8-dithiooctanoic acid) is a sulfur containing coenzyme essential for the activity of several key enzymes involved in oxidative and single carbon metabolism in most bacteria and eukaryotes. LA is synthetized by the concerted activity of the octanoyltransferase (LIP2, EC 2.3.1.181) and lipoyl synthase (LIP1, EC 2.8.1.8) enzymes. In plants, pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase or glycine decarboxylase are essential complexes that need to be lipoylated. These lipoylated enzymes and complexes are located in the mitochondria, while PDH is also present in plastids where it provides acetyl-CoA for de novo fatty acid biosynthesis. As such, lipoylation of PDH could regulate fatty acid synthesis in both these organelles. In the present work, the sunflower LIP1 and LIP2 genes (HaLIP1m and HaLIP2m) were isolated sequenced, cloned, and characterized, evaluating their putative mitochondrial location. The expression of these genes was studied in different tissues and protein docking was modeled. The genes were also expressed in Escherichia coli and Arabidopsis thaliana, where their impact on fatty acid and glycerolipid composition was assessed. Lipidomic studies in Arabidopsis revealed lipid remodeling in lines overexpressing these enzymes and the involvement of both sunflower proteins in the phenotypes observed is discussed in the light of the results obtained.
Highlights
Lipoic acid (LA; 6,8-dithiooctanoic acid) is a sulfur containing coenzyme found in most bacteria and eukaryotic organisms
This co-factor is essential for the activity of several key enzymes involved in oxidative and single carbon metabolism, including pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase (2-OGDH), branched-chain 2-oxoacid dehydrogenase (BCDH), Abbreviations: ACP, acyl carrier protein; BCDH, branched-chain 2-oxoacid dehydrogenase; DAF, days after flowering; DAG, diacylglycerol; FAMES, fatty acids methyl esters; FAS, fatty acid synthase; HaLIP1m, sunflower lipoyl synthase; HaLIP2m, sunflower octanoyltransferase; KAS, β-ketoacyl-(ACP) synthase; LA, lipoic acid; LIPA, bacterial lipoyl synthase; LIPB, bacterial octanoyltransferase; LIP1, plant lipoyl synthase; LIP2, plant octanoyltransferase; LPLA, lipoate protein ligase; 2-OGDH, 2-oxoglutarate dehydrogenase; PC, phosphatidylcholine; Principal Component Analysis (PCA), principal component analysis; PDH, pyruvate dehydrogenase; PE, phosphatidylethanolamine; SAM, S-adenosylmethionine; TAG, triacylglycerols; WT, wild type
Lipoic acid is generated from the octanoyl-acyl carrier protein (ACP) during de novo fatty acids biosynthesis through the activity of β-ketoacyl-(ACP) synthase (KAS)
Summary
Lipoic acid (LA; 6,8-dithiooctanoic acid) is a sulfur containing coenzyme found in most bacteria and eukaryotic organisms. The 2-OGDH and glycine cleavage system complexes are exclusive to mitochondria, whereas PDH is found in plastids where it is essential for de novo fatty acid biosynthesis (Martins-Noguerol et al, 2019) Both these organelles possess specific enzymatic machinery for LA biosynthesis and protein lipoylation. A different system for LA synthesis in sunflower was found, comprised of a lipoyl synthase (HaLIP1m) and an octanoyltransferase (HaLIP2m) that differed in their sequence and predicted location from the aforementioned plastidial forms Both genes were homologous to those previously described in A. thaliana mitochondria and they were both cloned, sequenced and characterized to study their expression and structure. The effect of the expression of these genes on fatty acid synthesis in E. coli and in transgenic Arabidopsis seeds, along with the description of the lipidomic remodeling in these transgenic seeds, illustrate the role of the mitochondrial lipoylation pathway in plant lipid metabolism
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