Lipoic acid attachment to proteins: stimulating new developments.

SUMMARYLipoic acid-modified proteins are essential for central metabolism and pathogenesis. In recent years, the Escherichia coli and Bacillus subtilis lipoyl assembly pathways have been modified and extended to archaea and diverse eukaryotes including humans. These extensions include a new pathway to insert the key sulfur atoms of lipoate, several new pathways of lipoate salvage, and a novel use of lipoic acid in sulfur-oxidizing bacteria. Other advances are the modification of E. coli LplA for studies of protein localization and protein-protein interactions in cell biology and in enzymatic removal of lipoate from lipoyl proteins. Finally, scenarios have been put forth for the evolution of lipoate assembly in archaea.

A novel IBA57 variant is associated with mitochondrial iron-sulfur protein deficiency and necrotizing myelopathy in dogs.

Introduction: Hereditary necrotizing myelopathy (HNM) in young Kooiker dogs is characterized by progressive ataxia and paralysis with autosomal recessive inheritance. The basic genetic defect is unknown. We investigated the possible cause by a genome-wide analysis using six affected and 17 unrelated unaffected Kooiker dogs and by functional follow-up studies. Method: The HNM locus was mapped by a case-control study using a dense SNP array and confirmed by linkage analysis of two pedigrees. The gene exons in the critical region were analyzed by next-generation sequencing. The functional effect of the candidate canine IBA57 pathogenic variant was biochemically examined in an established HeLa cell culture model in which the endogenous IBA75 gene product was depleted by RNAi. Results: The basic defect was localized in the centromeric 5 Mb region of canine chromosome 14. The most associated SNP co-segregated fully with HNM and reached an LOD score of 6.1. A candidate pathogenic mutation was found in the iron-sulfur cluster assembly gene IBA57 and led to the amino acid substitution R147W. The expression of human IBA57 harboring the canine R147W exchange could only partially restore the biochemical defects of several mitochondrial [4Fe-4S] proteins upon IBA57 depletion, showing that the mutant protein is functionally impaired. Discussion: Pathogenic variants in human IBA57 cause multiple mitochondrial dysfunction syndrome 3 (MMDS3), a neurodegenerative disorder with distant similarities to HNM. The incomplete functional complementation of IBA57-depleted human cells by IBA57-R147W identifies the DNA mutation in affected Kooiker dogs as the genetic cause of HNM. Our findings further expand the phenotypic spectrum of pathogenic IBA57 variants.

Assembly, transfer, and fate of mitochondrial iron-sulfur clusters.

Since the discovery of an autonomous iron-sulfur cluster (Fe-S) assembly machinery in mitochondria, significant efforts to examine the nature of this process have been made. The assembly of Fe-S clusters occurs in two distinct steps with the initial synthesis of [2Fe-2S] clusters by a first machinery followed by a subsequent assembly into [4Fe-4S] clusters by a second machinery. Despite this knowledge, we still have only a rudimentary understanding of how Fe-S clusters are transferred and distributed among their respective apoproteins. In particular, demand created by continuous protein turnover and the sacrificial destruction of clusters for synthesis of biotin and lipoic acid reveal possible bottlenecks in the supply chain of Fe-S clusters. Taking available information from other species into consideration, this review explores the mitochondrial assembly machinery of Arabidopsis and provides current knowledge about the respective transfer steps to apoproteins. Furthermore, this review highlights biotin synthase and lipoyl synthase, which both utilize Fe-S clusters as a sulfur source. After extraction of sulfur atoms from these clusters, the remains of the clusters probably fall apart, releasing sulfide as a highly toxic by-product. Immediate refixation through local cysteine biosynthesis is therefore an essential salvage pathway and emphasizes the physiological need for cysteine biosynthesis in plant mitochondria.

Metabolic engineering of Escherichia coli for high-level production of free lipoic acid.

L-Lipoic acid (LA) is an important antioxidant with various industrial applications as a nutraceutical and therapeutic. Currently, LA is produced by chemical synthesis. Cell factory development is complex as LA and its direct precursors only occur naturally in protein-bound forms. Here we report a rationally engineered LA cell factory and demonstrate de novo free LA production from glucose for the first time in E. coli. The pathway represents a significant challenge as the three key enzymes, native Octanoyltransferase (LipB) and Lipoyl Synthase (LipA), and heterologous Lipoamidase (LpA), are all toxic to overexpress in E. coli. To overcome the toxicity of LipB, functional metagenomic selection was used to identify a highly active and non-toxic LipB and LipA from S. liquefaciens. Using high throughput screening, we balanced translation initiation rates and dual, orthogonal induction systems for the toxic genes, LipA and LpA. The optimized strain yielded 2.5 mg free LA per gram of glucose in minimal media, expressing carefully balanced LipB and LipA, Enterococcus faecalis LpA, and a truncated, native, Dihydrolipoyllysine-residue acetyltransferase (AceF) lipoylation domain. When the optimized cell factory strain was cultivated in a fed-batch fermentation, a titer of 87 mg/L free LA in the supernatant was reached after 48 h. This titer is ∼3000-fold higher than previously reported free LA titer and ∼8-fold higher than the previous best total, protein-bound LA titer. The strategies presented here could be helpful in designing, constructing and balancing biosynthetic pathways that harbor toxic enzymes with protein-bound intermediates or products.

Mitochondrial [2Fe-2S] ferredoxins: new functions for old dogs.

Ferredoxins (FDXs) comprise a large family of iron-sulfur proteins that shuttle electrons from NADPH and FDX reductases into diverse biological processes. This review focuses on the structure, function and specificity of mitochondrial [2Fe-2S] FDXs that are related to bacterial FDXs due to their endosymbiotic inheritance. Their classical function in cytochrome P450-dependent steroid transformations was identified around 1960, and is exemplified by mammalian FDX1 (aka adrenodoxin). Thirty years later the essential function in cellular Fe/S protein biogenesis was discovered for the yeast mitochondrial FDX Yah1 that is additionally crucial for the formation of haem a and ubiquinone CoQ6 . In mammals, Fe/S protein biogenesis is exclusively performed by the FDX1 paralog FDX2, despite the high structural similarity of both proteins. Recently, additional and specific roles of human FDX1 in haem a and lipoyl cofactor biosyntheses were described. For lipoyl synthesis, FDX1 transfers electrons to the radical S-adenosyl methionine-dependent lipoyl synthase to kickstart its radical chain reaction. The high target specificity of the two mammalian FDXs is contained within small conserved sequence motifs, that upon swapping change the target selection of these electron donors.

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.

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.

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.

Solanum lycopersicum (tomato) possesses mitochondrial and plastidial lipoyl synthases capable of increasing lipoylation levels when expressed in bacteria.

Lipoic acid (LA) and its reduced form (dihydrolipoic acid, DHLA) have unique antioxidant properties among such molecules. Moreover, after a process termed lipoylation, LA is an essential prosthetic group covalently-attached to several key multi-subunit enzymatic complexes involved in primary metabolism, including E2 subunits of pyruvate dehydrogenase (PDH). The metabolic pathway of lipoylation has been extensively studied in Escherichia coli and Arabidopsis thaliana in which protein modification occurs via two routes: de novo synthesis and salvage. Common to both pathways, lipoyl synthase (LIP1 in plants, LipA in bacteria, EC 2.8.1.8) inserts sulphur atoms into the molecule in a final, activating step. However, despite the detection of LA and DHLA in other plant species, including tomato (Solanum lycopersicum), no plant LIP1s have been characterised to date from species other than Arabidopsis. In this work, we present the identification and characterisation of two LIPs from tomato, SlLIP1 and SlLIP1p. Consistent with in silico data, both are widely-expressed, particularly in reproductive organs. In line with bioinformatic predictions, we determine that yellow fluorescent protein tagged versions of SlLIP1 and SlLIP1p are mitochondrially- and plastidially-localised, respectively. Both possess the molecular hallmarks and domains of well-characterised bacterial LipAs. When heterologously-expressed in an E. coli lipA mutant, both are capable of complementing specific growth phenotypes and increasing lipoylation levels of E2 subunits of PDH in vivo, demonstrating that they do indeed function as lipoyl synthases.

Paramagnetic 1H NMR Spectroscopy to Investigate the Catalytic Mechanism of Radical S-Adenosylmethionine Enzymes.

Iron-sulfur clusters in radical S-adenosylmethionine (SAM) enzymes catalyze an astonishing array of complex and chemically challenging reactions across all domains of life. Here we showed that 1H NMR spectroscopy experiments tailored to reveal hyperfine-shifted signals of metal-ligands is a powerful tool to monitor the binding of SAM and of the octanoyl-peptide substrate to the two [4Fe-4S] clusters of human lipoyl synthase. The paramagnetically shifted signals of the iron-ligands were specifically assigned to each of the two bound [4Fe-4S] clusters, and then used to examine the interaction of SAM and substrate molecules with each of the two [4Fe-4S] clusters of human lipoyl synthase. 1H NMR spectroscopy can therefore contribute to the description of the catalityc mechanism of radical SAM enzymes.

Insight into the reaction mechanism of lipoyl synthase: a QM/MM study.

Lipoyl synthase (LipA) catalyses the final step of the biosynthesis of the lipoyl cofactor by insertion of two sulfur atoms at the C6 and C8 atoms of the protein-bound octanoyl substrate. In this reaction, two [4Fe4S] clusters and two molecules of S-adenosyl-L-methionine are used. One of the two FeS clusters is responsible for the generation of a powerful oxidant, the 5'-deoxyadenosyl radical (5'-dA•). The other (the auxiliary cluster) is the source of both sulfur atoms that are inserted into the substrate. In this paper, the spin state of the FeS clusters and the reaction mechanism is investigated by the combined quantum mechanical and molecular mechanics approach. The calculations show that the ground state of the two FeS clusters, both in the [4Fe4S]2+ oxidation state, is a singlet state with antiferromagnetically coupled high-spin Fe ions and that there is quite a large variation of the energies of the various broken-symmetry states, up to 40 kJ/mol. For the two S-insertion reactions, the highest energy barrier is found for the hydrogen-atom abstraction from the octanoyl substrate by 5'-dA•. The formation of 5'-dA• is very facile for LipA, with an energy barrier of 6 kJ/mol for the first S-insertion reaction and without any barrier for the second S-insertion reaction. In addition, the first S ion attack on the C6 radical of octanoyl was found to take place directly by the transfer of the H6 from the substrate to 5'-dA•, whereas for the second S-insertion reaction, a C8 radical intermediate was formed with a rate-limiting barrier of 71 kJ/mol.

Two redundant octanoyltransferases and one obligatory lipoyl synthase provide protein-lipoylation autonomy to plastids of Arabidopsis.

Octanoyltransferases (LIP2) are important for the lipoylation of several α-ketoacid decarboxylases and glycine decarboxylase, all of which are essential multienzyme complexes of central metabolism, by attaching de novo-synthesised octanoyl moieties to the respective target subunits. Lipoyl synthase (LIP1) then inserts two sulphur atoms each into the protein-bound octanoyl chains to generate the functional lipoamide arms. In plants, most of the above multienzyme complexes occur only in mitochondria. Pyruvate dehydrogenase is an exception, since it also occurs in plastids. Plastidial LIP1 and LIP2 are known, but it is not clear how essential these enzymes are. Here, we report that not just one but two redundant LIP2 isoforms, LIP2p and LIP2p2, operate in plastids of Arabidopsis. The combined deletion of the two isoenzymes is embryo-lethal. Deletion of the plastidial lipoyl synthase LIP1p is also embryo-lethal, indicating that all plastidial LIP1 activity is due to LIP1p. These features suggest that protein lipoylation is based on an autonomous and partially redundant de novo lipoylation pathway in plastids.