Characterization of Helianthus annuus Lipoic Acid Biosynthesis: The Mitochondrial Octanoyltransferase and Lipoyl Synthase Enzyme System

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.

The Fe-S cluster assembly factors NFU4 and NFU5 are primarily required for protein lipoylation in mitochondria

Plants have evolutionarily conserved NFU-domain proteins that are targeted to plastids or mitochondria. The 'plastid-type' NFU1, NFU2 and NFU3 in Arabidopsis thaliana play a role in iron-sulfur (Fe-S) cluster assembly in this organelle, whereas the type-II NFU4 and NFU5 proteins have not been subjected to mutant studies in any plant species to determine their biological role. Here we confirm that NFU4 and NFU5 are targeted to the mitochondria. The proteins are constitutively produced in all parts of the plant, suggesting a housekeeping function. Double nfu4 nfu5 knockout mutants were embryonic lethal, and depletion of the proteins led to growth arrest of young seedlings. Biochemical analyses revealed that NFU4 and NFU5 are required for lipoylation of the H proteins of the glycine decarboxylase complex and the E2 subunits of other mitochondrial dehydrogenases, with little impact on Fe-S cluster-containing respiratory complexes and aconitase. Consequently, the Gly-to-Ser ratio was increased in mutant seedlings and early growth was improved by elevated CO2. In addition, pyruvate, 2-oxoglutarate and branched-chain amino acids accumulated in the nfu4 nfu5 mutants, further supporting defects in the other three mitochondrial lipoate-dependent enzyme complexes. NFU4 and NFU5 interacted with mitochondrial lipoyl synthase (LIP1) in yeast 2-hybrid and bimolecular fluorescence complementation assays. These data indicate that NFU4 and NFU5 have a more specific function than previously thought, in providing Fe-S clusters to lipoyl synthase.

Characterization and Reconstitution of Human Lipoyl Synthase (LIAS) Supports ISCA2 and ISCU as Primary Cluster Donors and an Ordered Mechanism of Cluster Assembly

Lipoyl synthase (LIAS) is an iron–sulfur cluster protein and a member of the radical S-adenosylmethionine (SAM) superfamily that catalyzes the final step of lipoic acid biosynthesis. The enzyme contains two [4Fe–4S] centers (reducing and auxiliary clusters) that promote radical formation and sulfur transfer, respectively. Most information concerning LIAS and its mechanism has been determined from prokaryotic enzymes. Herein, we detail the expression, isolation, and characterization of human LIAS, its reactivity, and evaluation of natural iron–sulfur (Fe–S) cluster reconstitution mechanisms. Cluster donation by a number of possible cluster donor proteins and heterodimeric complexes has been evaluated. [2Fe–2S]-cluster-bound forms of human ISCU and ISCA2 were found capable of reconstituting human LIAS, such that complete product turnover was enabled for LIAS, as monitored via a liquid chromatography–mass spectrometry (LC–MS) assay. Electron paramagnetic resonance (EPR) studies of native LIAS and substituted derivatives that lacked the ability to bind one or the other of LIAS’s two [4Fe–4S] clusters revealed a likely order of cluster addition, with the auxiliary cluster preceding the reducing [4Fe–4S] center. These results detail the trafficking of Fe–S clusters in human cells and highlight differences with respect to bacterial LIAS analogs. Likely in vivo Fe–S cluster donors to LIAS are identified, with possible connections to human disease states, and a mechanistic ordering of [4Fe–4S] cluster reconstitution is evident.

A Structurally Novel Lipoyl Synthase in the Hyperthermophilic Archaeon Thermococcus kodakarensis

ABSTRACT Lipoic acid is a sulfur-containing cofactor and a component of the glycine cleavage system (GCS) involved in C1 compound metabolism and the 2-oxoacid dehydrogenases that catalyze the oxidative decarboxylation of 2-oxoacids. Lipoic acid is found in all domains of life and is generally synthesized as a lipoyl group on the H-protein of the GCS or the E2 subunit of 2-oxoacid dehydrogenases. Lipoyl synthase catalyzes the insertion of two sulfur atoms to the C-6 and C-8 carbon atoms of the octanoyl moiety on the octanoyl-H-protein or octanoyl-E2 subunit. Although the hyperthermophilic archaeon Thermococcus kodakarensis seemed able to synthesize lipoic acid, a classical lipoyl synthase (LipA) gene homolog cannot be found on the genome. In this study, we aimed to identify the lipoyl synthase in this organism. Genome information analysis suggested that the TK2109 and TK2248 genes, which had been annotated as biotin synthase (BioB), are both involved in lipoic acid metabolism. Based on the chemical reaction catalyzed by BioB, we predicted that the genes encode proteins that catalyze the lipoyl synthase reaction. Genetic analysis of TK2109 and TK2248 provided evidence that these genes are involved in lipoic acid biosynthesis. The purified TK2109 and TK2248 recombinant proteins exhibited lipoyl synthase activity toward a chemically synthesized octanoyl-octapeptide. These in vivo and in vitro analyses indicated that the TK2109 and TK2248 genes encode a structurally novel lipoyl synthase. TK2109 and TK2248 homologs are widely distributed among the archaeal genomes, suggesting that in addition to the LipA homologs, the two proteins represent a new group of lipoyl synthases in archaea. IMPORTANCE Lipoic acid is an essential cofactor for GCS and 2-oxoacid dehydrogenases, and α-lipoic acid has been utilized as a medicine and attracted attention as a supplement due to its antioxidant activity. The biosynthesis pathways of lipoic acid have been established in Bacteria and Eucarya but not in Archaea. Although some archaeal species, including Sulfolobus, possess a classical lipoyl synthase (LipA) gene homolog, many archaeal species, including T. kodakarensis, do not. In addition, the biosynthesis mechanism of the octanoyl moiety, a precursor for lipoyl group biosynthesis, is also unknown for many archaea. As the enzyme identified in T. kodakarensis most likely represents a new group of lipoyl synthases in Archaea, the results obtained in this study provide an important step in understanding how lipoic acid is synthesized in this domain and how the two structurally distinct lipoyl synthases evolved in nature.

Defining Caenorhabditis elegans as a model system to investigate lipoic acid metabolism

Lipoic acid (LA) is a sulfur-containing cofactor that covalently binds to a variety of cognate enzymes that are essential for redox reactions in all three domains of life. Inherited mutations in the enzymes that make LA, namely lipoyl synthase, octanoyltransferase, and amidotransferase, result in devastating human metabolic disorders. Unfortunately, because many aspects of this essential pathway are still obscure, available treatments only serve to alleviate symptoms. We envisioned that the development of an organismal model system might provide new opportunities to interrogate LA biochemistry, biology, and physiology. Here we report our investigations on three Caenorhabditis elegans orthologous proteins involved in this post-translational modification. We established that M01F1.3 is a lipoyl synthase, ZC410.7 an octanoyltransferase, and C45G3.3 an amidotransferase. Worms subjected to RNAi against M01F1.3 and ZC410.7 manifest larval arrest in the second generation. The arrest was not rescued by LA supplementation, indicating that endogenous synthesis of LA is essential for C. elegans development. Expression of the enzymes M01F1.3, ZC410.7, and C45G3.3 completely rescue bacterial or yeast mutants affected in different steps of the lipoylation pathway, indicating functional overlap. Thus, we demonstrate that, similarly to humans, C. elegans is able to synthesize LA de novo via a lipoyl-relay pathway, and suggest that this nematode could be a valuable model to dissect the role of protein mislipoylation and to develop new therapies.

Lipidomic Analysis of Plastidial Octanoyltransferase Mutants of Arabidopsis thaliana

Plant de novo fatty acid synthesis takes place in the plastid using acetyl-coenzyme A (acetyl-CoA) as the main precursor. This first intermediate is produced from pyruvate through the action of the plastidial pyruvate dehydrogenase complex (PDH), which catalyses the oxidative decarboxylation of pyruvate to produce acetyl-CoA, CO2, and NADH. For the proper functioning of this complex, lipoic acid is required to be bound to the dihydrolipoamide S-acetyltransferase E2 subunit of PDH. Octanoyltransferase (LIP2; EC 2.3.1.181) and lipoyl synthase (LIP1; EC 2.8.1.8) are the enzymes involved in the biosynthesis of this essential cofactor. In Arabidopsis plastids, an essential lipoyl synthase (AtLIP1p) and two redundant octanoyltransferases (AtLIP2p1 and AtLIP2p2) have been described. In the present study, the lipidomic characterization of Arabidopsis octanoyltransferase mutants reveals new insight into the lipoylation functions within plastid metabolism. Lipids and fatty acids from mature seeds and seedlings from Atlip2p1 and Atlip2p2 mutants were analysed by gas chromatography (GC) and liquid chromatography–electrospray ionization high-resolution mass spectrometry (LC-ESI-HRMS2), the analysis revealed changes in fatty acid profiles that showed similar patterns in both mutant seeds and seedlings and in the lipid species containing those fatty acids. Although both mutants showed similar tendencies, the lack of the AtLIP2p2 isoform produced a more acute variation in its lipids profile. These changes in fatty acid composition and the increase in their content per seed point to the interference of octanoyltransferases in the fatty acid synthesis flux in Arabidopsis thaliana seeds.

The A-type domain in Escherichia coli NfuA is required for regenerating the auxiliary [4Fe–4S] cluster in Escherichia coli lipoyl synthase

The lipoyl cofactor plays an integral role in several essential biological processes. The last step in its de novo biosynthetic pathway, the attachment of two sulfur atoms at C6 and C8 of an n-octanoyllysyl chain, is catalyzed by lipoyl synthase (LipA), a member of the radical SAM superfamily. In addition to the [4Fe–4S] cluster common to all radical SAM enzymes, LipA contains a second [4Fe–4S] auxiliary cluster, which is sacrificed during catalysis to supply the requisite sulfur atoms, rendering the protein inactive for further turnovers. Recently, it was shown that the Fe–S cluster carrier protein NfuA from Escherichia coli can regenerate the auxiliary cluster of E. coli LipA after each turnover, but the molecular mechanism is incompletely understood. Herein, using protein–protein interaction and kinetic assays as well as site-directed mutagenesis, we provide further insight into the mechanism of NfuA-mediated cluster regeneration. In particular, we show that the N-terminal A-type domain of E. coli NfuA is essential for its tight interaction with LipA. Further, we demonstrate that NfuA from Mycobacterium tuberculosis can also regenerate the auxiliary cluster of E. coli LipA. However, an Nfu protein from Staphylococcus aureus, which lacks the A-type domain, was severely diminished in facilitating cluster regeneration. Of note, addition of the N-terminal domain of E. coli NfuA to S. aureus Nfu, fully restored cluster-regenerating activity. These results expand our understanding of the newly discovered mechanism by which the auxiliary cluster of LipA is restored after each turnover.