Increased flexibility in the use of exogenous lipoic acid by Staphylococcus aureus

Lipoic acid is a cofactor required for intermediary metabolism that is either synthesized de novo or acquired from environmental sources. The bacterial pathogen Staphylococcus aureus encodes enzymes required for de novo biosynthesis, but also encodes two ligases, LplA1 and LplA2, that are sufficient for lipoic acid salvage during infection. S. aureus also encodes two H proteins, GcvH of the glycine cleavage system and the homologous GcvH‐L encoded in an operon with LplA2. GcvH is a recognized conduit for lipoyl transfer to α‐ketoacid dehydrogenase E2 subunits, while the function of GcvH‐L remains unclear. The potential to produce two ligases and two H proteins is an unusual characteristic of S. aureus that is unlike most other Gram positive Firmicutes and might allude to an expanded pathway of lipoic acid acquisition in this microorganism. Here, we demonstrate that LplA1 and LplA2 facilitate lipoic acid salvage by differentially targeting lipoyl domain‐containing proteins; LplA1 targets H proteins and LplA2 targets α‐ketoacid dehydrogenase E2 subunits. Furthermore, GcvH and GcvH‐L both facilitate lipoyl relay to E2 subunits. Altogether, these studies identify an expanded mode of lipoic acid salvage used by S. aureus and more broadly underscore the importance of bacterial adaptations when faced with nutritional limitation.     

LplA1-dependent utilization of host lipoyl peptides enables Listeria cytosolic growth and virulence

The bacterial pathogen Listeria monocytogenes replicates within the cytosol of mammalian cells. Mechanisms by which the bacterium exploits the host cytosolic environment for essential nutrients are poorly defined. L. monocytogenes is a lipoate auxotroph and must scavenge this critical cofactor, using lipoate ligases to facilitate attachment of the lipoyl moiety to metabolic enzyme complexes. Although the L. monocytogenes genome encodes two putative lipoate ligases, LplA1 and LplA2, intracellular replication and virulence require only LplA1. Here we show that LplA1 enables utilization of host-derived lipoyl peptides by L. monocytogenes. LplA1 is dispensable for growth in the presence of free lipoate, but necessary for growth on low concentrations of mammalian lipoyl peptides. Furthermore, we demonstrate that the intracellular growth defect of the DeltalplA1 mutant is rescued by addition of exogenous lipoic acid to host cells, suggesting that L. monocytogenes dependence on LplA1 is dictated by limiting concentrations of available host lipoyl substrates. Thus, the ability of L. monocytogenes and other intracellular pathogens to efficiently use host lipoyl peptides as a source of lipoate may be a requisite adaptation for life within the mammalian cell.     

Lipoic Acid Synthesis and Attachment in Yeast Mitochondria

Lipoic acid is a sulfur-containing cofactor required for the function of several multienzyme complexes involved in the oxidative decarboxylation of α-keto acids and glycine. Mechanistic details of lipoic acid metabolism are unclear in eukaryotes, despite two well defined pathways for synthesis and covalent attachment of lipoic acid in prokaryotes. We report here the involvement of four genes in the synthesis and attachment of lipoic acid in Saccharomyces cerevisiae. LIP2 and LIP5 are required for lipoylation of all three mitochondrial target proteins: Lat1 and Kgd2, the respective E2 subunits of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, and Gcv3, the H protein of the glycine cleavage enzyme. LIP3, which encodes a lipoate-protein ligase homolog, is necessary for lipoylation of Lat1 and Kgd2, and the enzymatic activity of Lip3 is essential for this function. Finally, GCV3, encoding the H protein target of lipoylation, is itself absolutely required for lipoylation of Lat1 and Kgd2. We show that lipoylated Gcv3, and not glycine cleavage activity per se, is responsible for this function. Demonstration that a target of lipoylation is required for lipoylation is a novel result. Through analysis of the role of these genes in protein lipoylation, we conclude that only one pathway for de novo synthesis and attachment of lipoic acid exists in yeast. We propose a model for protein lipoylation in which Lip2, Lip3, Lip5, and Gcv3 function in a complex, which may be regulated by the availability of acetyl-CoA, and which in turn may regulate mitochondrial gene expression.Several oxidative decarboxylation reactions are carried out in prokaryotes and eukaryotes by multienzyme complexes. The function of these complexes requires the action of a sulfur-containing cofactor, lipoic acid (6,8-thioctic acid) (1, 2). Lipoic acid is covalently attached via an amide linkage to a specific lysine residue on the surface of the conserved lipoyl domain of the E2 subunits of pyruvate dehydrogenase (PDH),³ α-ketoglutarate dehydrogenase (α-KDH), the branched chain α-keto acid dehydrogenase complexes, and the H protein of the glycine cleavage (GC) enzyme (3). The lipoyl moiety serves as a swinging arm that shuttles reaction intermediates between active sites within the complexes (1). Despite the well characterized function of lipoic acid as a prosthetic group, the mechanisms of its synthesis and attachment to proteins are the subject of ongoing investigations (4–7).These reactions are best understood in Escherichia coli, which has two well defined pathways for lipoic acid synthesis and attachment: a de novo pathway and a salvage pathway (8). Octanoic acid, synthesized on the acyl carrier protein (ACP) (9), is the substrate for the de novo pathway. Lipoyl synthase (LipA) catalyzes the addition of two sulfur atoms to form lipoic acid from octanoic acid either before or after transfer to the target protein (10) by lipoyl(octanoyl)-ACP:protein transferase (LipB) (11, 12). The preferred order of these two reactions is attachment of octanoic acid by LipB, followed by addition of sulfur by LipA (13). By contrast, in the salvage pathway, lipoate-protein ligase (LplA) attaches free lipoic acid to proteins in a two-step reaction. Lipoic acid, which can be scavenged from the medium, is first activated to lipoyl-AMP and then the lipoyl group is transferred to the proteins (14).Lipoic acid synthesis and attachment to target proteins are less well understood in eukaryotes. Homologs of the E. coli enzymes have been found in fungi, plants, protists, and mammals, but many mechanistic details are unclear (15–17). In Saccharomyces cerevisiae, the mitochondrial type II fatty acid biosynthetic pathway (FAS II) synthesizes octanoyl-ACP, which is the substrate for de novo lipoic acid synthesis (18). Lip2 and Lip5, the respective yeast homologs of E. coli LipB and LipA, were shown to be required for respiratory growth on glycerol medium, PDH activity (19), and lipoic acid synthesis (20), indicating functional roles in de novo lipoic acid synthesis and attachment. However, there has been no previous report of an LplA-like lipoate-protein ligase homolog in yeast. Furthermore, lip2 and lip5 mutant strains cannot grow on medium containing lipoic acid (19, 20), suggesting that yeast either cannot use exogenously supplied lipoic acid or there is no yeast equivalent of the E. coli LplA-driven salvage pathway.Here we report the involvement of two additional enzymes in protein lipoylation in yeast mitochondria. The first, Lip3, is a lipoate-protein ligase homolog and is required with Lip2 and Lip5 for lipoylation of the E2 subunits of PDH (Lat1) and α-KDH (Kgd2). The second enzyme, Gcv3, the H protein of the GC enzyme, is absolutely required for lipoylation of all proteins in yeast.     

Knockout Studies Reveal an Important Role of Plasmodium Lipoic Acid Protein Ligase A1 for Asexual Blood Stage Parasite Survival

Lipoic acid (LA) is a dithiol-containing cofactor that is essential for the function of α-keto acid dehydrogenase complexes. LA acts as a reversible acyl group acceptor and ‘swinging arm’ during acyl-coenzyme A formation. The cofactor is post-translationally attached to the acyl-transferase subunits of the multienzyme complexes through the action of octanoyl (lipoyl): N-octanoyl (lipoyl) transferase (LipB) or lipoic acid protein ligases (LplA). Remarkably, apicomplexan parasites possess LA biosynthesis as well as scavenging pathways and the two pathways are distributed between mitochondrion and a vestigial organelle, the apicoplast. The apicoplast-specific LipB is dispensable for parasite growth due to functional redundancy of the parasite''s lipoic acid/octanoic acid ligases/transferases. In this study, we show that LplA1 plays a pivotal role during the development of the erythrocytic stages of the malaria parasite. Gene disruptions in the human malaria parasite P. falciparum consistently were unsuccessful while in the rodent malaria model parasite P. berghei the LplA1 gene locus was targeted by knock-in and knockout constructs. However, the LplA1⁽⁻⁾ mutant could not be cloned suggesting a critical role of LplA1 for asexual parasite growth in vitro and in vivo. These experimental genetics data suggest that lipoylation during expansion in red blood cells largely occurs through salvage from the host erythrocytes and subsequent ligation of LA to the target proteins of the malaria parasite.     

Crystal structure of lipoate-protein ligase A bound with the activated intermediate: insights into interaction with lipoyl domains

Lipoic acid is the covalently attached cofactor of several multi-component enzyme complexes that catalyze key metabolic reactions. Attachment of lipoic acid to the lipoyl-dependent enzymes is catalyzed by lipoate-protein ligases (LPLs). In Escherichia coli, two distinct enzymes lipoate-protein ligase A (LplA) and lipB-encoded lipoyltransferase (LipB) catalyze independent pathways for lipoylation of the target proteins. The reaction catalyzed by LplA occurs in two steps. First, LplA activates exogenously supplied lipoic acid at the expense of ATP to lipoyl-AMP. Next, it transfers the enzyme-bound lipoyl-AMP to the epsilon-amino group of a specific lysine residue of the lipoyl domain to give an amide linkage. To gain insight into the mechanism of action by LplA, we have determined the crystal structure of Thermoplasma acidophilum LplA in three forms: (i) the apo form; (ii) the ATP complex; and (iii) the lipoyl-AMP complex. The overall fold of LplA bears some resemblance to that of the biotinyl protein ligase module of the E. coli biotin holoenzyme synthetase/bio repressor (BirA). Lipoyl-AMP is bound deeply in the bifurcated pocket of LplA and adopts a U-shaped conformation. Only the phosphate group and part of the ribose sugar of lipoyl-AMP are accessible from the bulk solvent through a tunnel-like passage, whereas the rest of the activated intermediate is completely buried inside the active site pocket. This first view of the activated intermediate bound to LplA allowed us to propose a model of the complexes between Ta LplA and lipoyl domains, thus shedding light on the target protein/lysine residue specificity of LplA.     

Protein-protein interactions in assembly of lipoic acid on the 2-oxoacid dehydrogenases of aerobic metabolism

Lipoic acid is a covalently attached cofactor essential for the activity of 2-oxoacid dehydrogenases and the glycine cleavage system. In the absence of lipoic acid modification, the dehydrogenases are inactive, and aerobic metabolism is blocked. In Escherichia coli, two pathways for the attachment of lipoic acid exist, a de novo biosynthetic pathway dependent on the activities of the LipB and LipA proteins and a lipoic acid scavenging pathway catalyzed by the LplA protein. LipB is responsible for octanoylation of the E2 components of 2-oxoacid dehydrogenases to provide the substrates of LipA, an S-adenosyl-L-methionine radical enzyme that inserts two sulfur atoms into the octanoyl moiety to give the active lipoylated dehydrogenase complexes. We report that the intact pyruvate and 2-oxoglutarate dehydrogenase complexes specifically copurify with both LipB and LipA. Proteomic, genetic, and dehydrogenase activity data indicate that all of the 2-oxoacid dehydrogenase components are present. In contrast, LplA, the lipoate protein ligase enzyme of lipoate salvage, shows no interaction with the 2-oxoacid dehydrogenases. The interaction is specific to the dehydrogenases in that the third lipoic acid-requiring enzyme of Escherichia coli, the glycine cleavage system H protein, does not copurify with either LipA or LipB. Studies of LipB interaction with engineered variants of the E2 subunit of 2-oxoglutarate dehydrogenase indicate that binding sites for LipB reside both in the lipoyl domain and catalytic core sequences. We also report that LipB forms a very tight, albeit noncovalent, complex with acyl carrier protein. These results indicate that lipoic acid is not only assembled on the dehydrogenase lipoyl domains but that the enzymes that catalyze the assembly are also present "on site."     

Unsaturated Long Chain Free Fatty Acids Are Input Signals of the Salmonella enterica PhoP/PhoQ Regulatory System

The Salmonella enterica serovar Typhimurium PhoP/PhoQ system has largely been studied as a paradigmatic two-component regulatory system not only to dissect structural and functional aspects of signal transduction in bacteria but also to gain knowledge about the versatile devices that have evolved allowing a pathogenic bacterium to adjust to or counteract environmental stressful conditions along its life cycle. Mg²⁺ limitation, acidic pH, and the presence of cationic antimicrobial peptides have been identified as cues that the sensor protein PhoQ can monitor to reprogram Salmonella gene expression to cope with extra- or intracellular challenging conditions. In this work, we show for the first time that long chain unsaturated free fatty acids (LCUFAs) present in Salmonella growth medium are signals specifically detected by PhoQ. We demonstrate that LCUFAs inhibit PhoQ autokinase activity, turning off the expression of the PhoP-dependent regulon. We also show that LCUFAs exert their action independently of their cellular uptake and metabolic utilization by means of the β-oxidative pathway. Our findings put forth the complexity of input signals that can converge to finely tune the activity of the PhoP/PhoQ system. In addition, they provide a new potential biochemical platform for the development of antibacterial strategies to fight against Salmonella infections.     

The Thermoplasma acidophilum LplA-LplB Complex Defines a New Class of Bipartite Lipoate-protein Ligases

Lipoic acid is a covalently bound cofactor found throughout the domains of life that is required for aerobic metabolism of 2-oxoacids and for C₁ metabolism. Utilization of exogenous lipoate is catalyzed by a ligation reaction that proceeds via a lipoyl-adenylate intermediate to attach the cofactor to the ϵ-amino group of a conserved lysine residue of protein lipoyl domains. The lipoyl ligases of demonstrated function have a large N-terminal catalytic domain and a small C-terminal accessory domain. Half of the members of the LplA family detected in silico have only the large catalytic domain. Two x-ray structures of the Thermoplasma acidophilum LplA structure have been reported, although the protein was reported to lack ligase activity. McManus et al. (McManus, E., Luisi, B. F., and Perham, R. N. (2006) J. Mol. Biol. 356, 625–637) hypothesized that the product of an adjacent gene was also required for ligase activity. We have shown this to be the case and have named the second protein, LplB. We found that complementation of Escherichia coli strains lacking lipoate ligase with T. acidophilum LplA was possible only when LplB was also present. LplA had no detectable ligase activity in vitro in the absence of LplB. Moreover LplA and LplB were shown to interact and were purified as a heterodimer. LplB was required for lipoyl-adenylate formation but was not required for transfer of the lipoyl moiety of lipoyl-adenylate to acceptor proteins. Surveys of sequenced genomes show that most lipoyl ligases of the kingdom Archaea are heterodimeric. We propose that the presence of an accessory domain provides a diagnostic to distinguish lipoyl ligase homologues from other members of the lipoate/biotin attachment enzyme family.Lipoic acid is a covalently bound cofactor that conveys activated reaction intermediates between different active sites of multienzyme complexes (1). Lipoate is essential for aerobic metabolism of 2-oxoacids and for glycine cleavage. In its active form lipoate is attached to the ϵ-amino group of a small (∼80-residue) well conserved lipoyl domain (LD)² lysine residue via an amide bond. LDs are typically found at the N termini of the E2 subunits of 2-oxoacid dehydrogenase complexes. In the 2-oxoacid complexes, lipoylated LD receives the decarboxylated acid from the E1 subunit active site in thioester linkage to a lipoate thiol. The acyl thioester is then converted to the corresponding CoA thioester by thioester exchange catalyzed by the E2 subunit active site. The dihydrolipoamide dehydrogenase subunit (E3) then oxidizes the dihydrolipoyl-LD back to the lipoyl-LD to reset the catalytic cycle. In the glycine cleavage (also called glycine decarboxylase and glycine dehydrogenase) system, the lipoyl domain exists as a free protein designated H. Lipoyl-LD receives the product of glycine decarboxylation, methylamine, from the P protein. The methylamine is then transferred to the T protein to produce methylenetetrahydrofolate that is typically used to synthesize serine from a second molecule of glycine. In Escherichia coli, lipoic acid is essential for aerobic growth because of the need for pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. The glycine cleavage system is not required for growth of wild type E. coli strains but is required for growth of Arabidopsis where the H protein can be present at millimolar concentrations in photosynthetic cells (2).The reactions whereby lipoic acid-modified proteins are produced are best understood in E. coli. The most straightforward pathway is via lipoate-protein ligase, an activity first described by L. J. Reed et al. (3) (see Fig. 1). These workers postulated that lipoate was attached to protein by a two-step ATP-dependent reaction with lipoyl-AMP as an activated intermediate (Fig. 1). Although the lipoate-protein ligases were key reagents in demonstration of the role of lipoic acid in the 2-oxoacid dehydrogenase reactions (3, 4), the protein was not purified to homogeneity, and thus the proposed mechanism could not be considered proved. The E. coli lplA gene was the first gene encoding a lipoate-protein ligase isolated, and LplA was the first such ligase purified to homogeneity (5, 6). The isolation of null mutants in lplA showed that LplA does not play a role in de novo lipoic acid synthesis but rather acts to scavenge lipoic acid from the environment (6, 7).Open in a separate windowFIGURE 1.Lipoic acid metabolism in E. coli. Panel A, the lipoyl ligase (LplA) reaction that proceeds through the lipoyl-adenylate intermediate. In E. coli LplA acts to scavenge lipoic acid from the growth medium. Panel B, schematic of lipoic acid synthesis in E. coli. LipB transfers an octanoyl moiety from the fatty acid biosynthetic intermediate, octanoyl-acyl carrier protein, to the LD domain of a lipoate-accepting protein (in this case the E2 subunit of a 2-oxoacid dehydrogenase). The octanoylated LD domain is the substrate of LipA, an S-adenosylmethionine radical enzyme that replaces one hydrogen atom on each of octanoate carbons 6 and 8 with sulfur atoms. Panel C, the differing arrangements of genes and domains found in lipoate ligases in T. acidophilum, E. coli, and Streptomyces coelicolor. Only a single nucleotide lies between the T. acidophilum LplB and LplA coding sequences.LplA is a 38-kDa monomeric protein (5). Assays with a fully defined system have demonstrated that LplA plus lipoate and Mg-ATP are sufficient to reconstitute lipoylation in vitro and that lipoyl-AMP is a reaction intermediate (5, 6, 8, 9). Thus, it is clear that LplA catalyzes both the ATP-dependent activation of lipoate to lipoyl-AMP as well as the transfer of this activated lipoyl species to apoprotein with concomitant release of AMP. The E. coli LplA enzyme has been shown to be capable of utilizing lipoate and several lipoate analogues such as octanoate as donors for the post-translational modification of E2 apoproteins in vivo (5, 6).Recently crystal structures of E. coli LplA and of LplA homologues have been reported including an E. coli LplA-lipoic acid complex (10–12). The reported structures of the unliganded proteins agree well and show E. coli LplA to be a two-domain protein consisting of a large N-terminal domain and a small C-terminal domain (Figs. 1 and ​and2).2). However, the E. coli LplA-lipoic acid complex is difficult to interpret because lipoic acid molecules were heterogeneously bound to LplA molecules within the crystals and were poorly resolved. In one case the lipoic acid carboxyl was hydrogen-bonded to Ser-72, whereas in another case Arg-140 was the hydrogen bond donor (10). Because enzymes rarely show such plasticity and lipoic acid is a hydrophobic molecule, it seemed possible that the observed association of the cofactor with a hydrophobic LplA surface in the interdomain cavity was artifactual. Moreover in prior work K. E. Reed et al. (13) had isolated LplA mutants resistant to inhibition by an analogue of lipoic acid in which the sulfur atoms had been replaced with selenium. Because this is a very discrete modification of the LplA substrate, the mutant protein would be expected to have an alteration close to the pocket that binds the lipoic acid thiolane ring. However, the site of this mutation (Gly-76 to serine (7)) was distal from the lipoate binding site reported. This dilemma was resolved by two lipoic acid-containing structures of an LplA homologue from the archaeon Thermoplasma acidophilum (11, 12) that can be readily superimposed on the E. coli LplA structure except that the T. acidophilum protein lacks the E. coli LplA C-terminal domain (Fig. 2). In both T. acidophilum structures the lipoate thiolane ring was adjacent to the glycine residue that corresponds to E. coli Gly-76, the residue giving resistance to the selenium analogue, and a plausible reorganization of the molecule to prevent binding of the slightly larger analogue was proposed (12). Moreover addition of lipoic acid to a complex of the T. acidophilum LplA with ATP gave lipoyl-AMP showing that the lipoic acid was bound in a physiologically meaningful manner (11). The lipoyl-AMP was bound in a U-shaped pocket and was well shielded from solvent. Thus, it seems that the locations of the lipoate moieties in the two T. acidophilum LplA structures indicate that these represent catalytically competent lipoate binding sites (rather than the sites of E. coli LplA where lipoate bound). A caveat was that the T. acidophilum LplA was inactive in catalysis of the overall LplA reaction (12). Because T. acidophilum LplA lacks the C-terminal domain (CTD) of E. coli LplA (11, 12), this suggested that the missing domain was required for activity, and a second protein was proposed to interact with T. acidophilum LplA to allow the complete reaction (12). If this were the case, the T. acidophilum lipoyl ligase would provide an unusually facile system to investigate the role of the CTD in lipoate-protein ligases.Open in a separate windowFIGURE 2.Structural alignments of LplA and LipB structures. Previously published crystal structures were aligned using DeepView (37). Panel A, E. coli LplA (Protein Data Bank code 1X2H in green) aligned with T. acidophilum LplA (Protein Data Bank code 2ART in orange). The lipoyl-adenylate intermediate bound to T. acidophilum LplA is shown in purple. The adenylate binding loop is indicated with an arrow. Panel B, M. tuberculosis LipB (Protein Data Bank code 1W66 in gray) is aligned with the E. coli LplA structure of panel A. The purple line denotes the covalent decanoate adduct present in the M. tuberculosis LipB structure. The substrate binding pocket is conserved among members of the protein family. The accessory domain is not part of the binding pocket and appears to play an indirect role in catalysis.If the lipoate-protein ligase reaction can be catalyzed by a heteromeric protein this may allow better discrimination of lipoyl ligases from acyl carrier protein:protein octanoyltransferases. In E. coli de novo lipoic acid biosynthesis is accomplished by two enzymes, the LipB octanoyltransferase and the LipA lipoyl synthase (14) (Fig. 1). LipB transfers the octanoate moiety from the octanoyl-acyl carrier protein intermediate of fatty acid biosynthesis to the ϵ-amine of the conserved LD lysine residue resulting in amide-linked octanoate (Fig. 1). LipA then catalyzes replacement of a hydrogen atom on each of octanoate carbons 6 and 8 with sulfur atoms derived from a LipA iron-sulfur center via an S-adenosylmethionine-dependent radical mechanism (14, 15). That is, lipoic acid is assembled on its cognate proteins (16).Although the two classes of LD-modifying enzymes, LplA and LipB, show very low amino acid sequence conservation and utilize different chemistries, the proteins surprisingly show structural conservation and have related active site architectures (17, 18) (Fig. 2). The Mycobacterium tuberculosis LipB and T. acidophilum LplA can be superimposed by using all matching Cα positions with a root mean square deviation of ≈2.5 Å with good topological matching of most secondary structural elements (18). Hence in length and structure LipBs resemble LplAs that lack the C-terminal domain. Although the E. coli LipB and LplA sequences align very poorly, a large number of proteins in the data bases have similarities to both proteins, and therefore annotation of a given protein as a ligase or octanoyltransferase is not straightforward. If an LplA CTD can be a separate protein, an additional criterion to distinguish lipoate ligases and octanoyltransferases would be available. It should be noted that biotin ligases also show structural (but not sequence) conservation with LipB and LplA, and this group of proteins comprises the Pfam family PF03099 (19). However, all known biotin ligases have a C-terminal domain that greatly aids in their annotation. We report that, as predicted by McManus et al. (12), the CTD function essential for lipoate-protein ligase activity is encoded by a gene located immediately upstream of T. acidophilum lplA that we call lplB.     

Kinetic analysis of the role of lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

The catalytic roles of the two reductively acetylatable lipoic acid residues on each lipoate acetyltransferase chain of the pyruvate dehydrogenase complex of Escherichia coli were investigated. Both lipoyl groups are reductively acetylated from pyruvate at the same apparent rate and both can transfer their acetyl groups to CoASH, part-reactions of the overall complex reaction. The complex was treated with N-ethylmaleimide in the presence of pyruvate and the absence of CoASH, conditions that lead to the modification and inactivation of the S-acetyldihydrolipoic acid residues. Modification was found to proceed appreciably faster than the accompanying loss of enzymic activity. The kinetics of the modification were fitted best by supposing that the two lipoyl groups react with the maleimide at different rates, one being modified at approximately 3.5 times the rate of the other. The loss of complex activity took place at a rate approximately equal to that calculated for the modification of the more slowly reacting lipoic acid residue. The simplest interpretation of this result is that only this residue is essential in the overall catalytic mechanism, but an alternative explanation in which one lipoic acid residue can take over the function of another was not ruled out. The kinetics of inactivation could not be reconciled with an obligatory serial interaction between the two lipoic acid residues. Similar experiments with the fluorescent N-[p-(benzimidazol-2-yl)phenyl]maleimide supported these conclusions, although the modification was found to be less specific than with N-ethylmaleimide. The more rapidly modified lipoic acid residue may be involved in the system of intramolecular transacetylation reactions that couple active sites in the lipoate acetyltransferase component.     

Scavenging of Cytosolic Octanoic Acid by Mutant LplA Lipoate Ligases Allows Growth of Escherichia coli Strains Lacking the LipB Octanoyltransferase of Lipoic Acid Synthesis

The LipB octanoyltransferase catalyzes the first step of lipoic acid synthesis in Escherichia coli, transfer of the octanoyl moiety from octanoyl-acyl carrier protein to the lipoyl domains of the E2 subunits of the 2-oxoacid dehydrogenases of aerobic metabolism. Strains containing null mutations in lipB are auxotrophic for either lipoic acid or octanoic acid. We report the isolation of two spontaneously arising mutant strains that allow growth of lipB strains on glucose minimal medium; we determined that suppression was caused by single missense mutations within the coding sequence of the gene (lplA) that encodes lipoate-protein ligase. The LplA proteins encoded by the mutant genes have reduced K_m values for free octanoic acid and thus are able to scavenge cytosolic octanoic acid for octanoylation of lipoyl domains.Escherichia coli has three lipoic acid-dependent enzyme systems: pyruvate dehydrogenase (PDH), 2-oxoglutarate dehydrogenase (OGDH), and the glycine cleavage system (GCV) (8). PDH catalyzes the oxidative decarboxylation of pyruvate to acetyl-coenzyme A (CoA), the tricarboxylic acid (TCA) cycle substrate and fatty acid building block. OGDH functions in the TCA cycle, where it catalyzes the decarboxylation of 2-oxoglutarate to succinyl-CoA, the precursor of several amino acids. GCV is involved in the breakdown of glycine into ammonia and C₁ units. Whereas GCV is expressed only in the presence of glycine, PDH and OGDH are required for aerobic growth. (During anaerobic growth, acetyl-CoA is synthesized by other enzymes and an OGDH-independent branched form of the TCA cycle forms succinyl-CoA from succinate.) The three enzyme systems contain subunits (the E2 subunits of PDH and OGDH and the H protein of GCV) which contain at least one lipoyl domain, a conserved structure of ca. 80 residues (8). Lipoic acid is attached in an amide bond to a specific lysine residue of these domains, where it functions as a classical “swinging arm,” carrying reaction intermediates between the active sites of the lipoate-dependent systems (27).Lipoic acid [R-5-(1,2-dithiolan-3-yl)pentanoic acid, also called 6,8-dithiooctanoic acid and thioctic acid] is composed of an eight-carbon fatty acid backbone to which two sulfur atoms are attached at carbons 6 and 8 (Fig. ​(Fig.1).1). In the oxidized state, the sulfur atoms are in a disulfide linkage forming a five-membered ring with three backbone carbons. The disulfide bond is reduced upon binding of the intermediates (an acetyl moiety in the case of PDH, a succinyl moiety in the case of OGDH, and an aminomethyl moiety in the case of GCV). Following release of the intermediates to form the products of the enzyme complexes, the reduced lipoyl moiety must be reoxidized before entering another catalytic cycle. Oxidation is catalyzed by lipoamide dehydrogenase, a subunit component of the three lipoic acid-dependent enzyme systems (8). E. coli strains defective in lipoic acid biosynthesis are unable to grow on aerobic glucose minimal media unless the media are supplemented with acetate and succinate to bypass the need for the two lipoic acid-dependent dehydrogenases (15, 32).Open in a separate windowFIG. 1.Lipoic acid metabolism in E. coli. (A) LplA lipoate ligase reaction, in which lipoate reacts with ATP to form the activated intermediate, lipoyl-adenylate (lipoyl-AMP), which remains firmly bound within the active site. The lipoyl-adenylate mixed anhydride bond is then attacked by the ɛ-amino group of the target lysine residue of the acceptor lipoyl domain to form lipoylated protein. LplA also utilizes octanoic acid. (B) Lipoic acid synthesis in E. coli. LipB transfers an octanoyl moiety from the fatty acid biosynthetic intermediate, octanoyl-ACP, to the lipoyl domain of a lipoate-accepting protein (in this case the E2 subunit of a 2-oxoacid dehydrogenase). The octanoylated domain is the substrate of LipA, an S-adenosylmethionine radical enzyme that replaces one hydrogen atom on each of octanoate carbons 6 and 8 with sulfur atoms. For a review, see reference 8.Studies in our laboratory and others have elucidated the lipoic acid synthesis pathway of E. coli (Fig. ​(Fig.1).1). The LipB octanoyl-[acyl carrier protein {ACP}]:protein N-octanoyltransferase (20, 33, 34) transfers the octanoyl moiety from octanoyl-ACP, a fatty acid biosynthetic intermediate, to lipoyl domains. This reaction proceeds through an acyl enzyme intermediate in which the octanoyl moiety is in thioester linkage to a conserved cysteine residue in the enzyme active site (22, 33). The thioester bond is then attacked by the ɛ-amino group of the target lipoyl domain lysine residue to give the amide-linked lipoate moiety. The product of this catalysis, an octanoyl domain, is the substrate of the LipA lipoate synthase, an S-adenosylmethionine radical enzyme which inserts sulfur atoms at carbons 6 and 8. In addition to the LipB-LipA pathway of lipoic acid synthesis, E. coli also contains an enzyme that scavenges lipoic acid from the growth medium, the LplA lipoate-protein ligase. LplA uses ATP to activate lipoic acid to lipoyl-adenylate, the mixed anhydride of which is attacked by the lipoyl domain lysine reside to give the lipoylated domain (Fig. ​(Fig.1).1). LplA is also active with octanoic acid and efficiently attaches exogenous octanoate to lipoyl domains both in vivo and in vitro (11, 25, 26, 34). lplA null mutants have no phenotype in strains having an intact lipoic acid synthesis pathway (26).The subject of this report is the behavior of lipB null mutants, which (as expected from the above discussion) are lipoic acid auxotrophs (26, 32). Growth of lipB strains can also be supported by supplementation of the medium with octanoate (34). Upon plating of lipB null mutants on plates of minimal glucose medium, colonies arise that no longer require lipoic acid (26). These are suppressor mutations because the block in lipoic acid synthesis remains. Suppression in the strains studied in this work maps to the lplA gene. The LplA proteins encoded by these suppressor mutants contain point mutations that greatly decrease the Michaelis constant for free octanoic acid and allow efficient scavenging of cytosolic octanoate.     

A novel two-gene requirement for the octanoyltransfer reaction of Bacillus subtilis lipoic acid biosynthesis

The Bacillus subtilis genome encodes three apparent lipoyl ligase homologues: yhfJ, yqhM and ywfL, which we have renamed lplJ, lipM and lipL respectively. We show that LplJ encodes the sole lipoyl ligase of this bacterium. Physiological and biochemical characterization of a ΔlipM strain showed that LipM is absolutely required for the endogenous lipoylation of all lipoate-dependent proteins, confirming its role as the B. subtilis octanoyltransferase. However, we also report that in contrast to Escherichia coli, B. subtilis requires a third protein for lipoic acid assembly, LipL. B. subtilis ΔlipL strains are unable to synthesize lipoic acid despite the presence of LipM and the sulphur insertion enzyme, LipA, which should suffice for lipoic acid biosynthesis based on the E. coli model. LipM is only required for the endogenous lipoylation pathway, whereas LipL also plays a role in lipoic acid scavenging. Expression of E. coli lipB allows growth of B. subtilisΔlipL or ΔlipM strains in the absence of supplements. In contrast, growth of an E. coliΔlipB strain can be complemented with lipM, but not lipL. These data together with those of the companion article provide evidence that LipM and LipL catalyse sequential reactions in a novel pathway for lipoic acid biosynthesis.     

Comparative Genomic Analysis Reveals 2-Oxoacid Dehydrogenase Complex Lipoylation Correlation with Aerobiosis in Archaea

Metagenomic analyses have advanced our understanding of ecological microbial diversity, but to what extent can metagenomic data be used to predict the metabolic capacity of difficult-to-study organisms and their abiotic environmental interactions? We tackle this question, using a comparative genomic approach, by considering the molecular basis of aerobiosis within archaea. Lipoylation, the covalent attachment of lipoic acid to 2-oxoacid dehydrogenase multienzyme complexes (OADHCs), is essential for metabolism in aerobic bacteria and eukarya. Lipoylation is catalysed either by lipoate protein ligase (LplA), which in archaea is typically encoded by two genes (LplA-N and LplA-C), or by a lipoyl(octanoyl) transferase (LipB or LipM) plus a lipoic acid synthetase (LipA). Does the genomic presence of lipoylation and OADHC genes across archaea from diverse habitats correlate with aerobiosis? First, analyses of 11,826 biotin protein ligase (BPL)-LplA-LipB transferase family members and 147 archaeal genomes identified 85 species with lipoylation capabilities and provided support for multiple ancestral acquisitions of lipoylation pathways during archaeal evolution. Second, with the exception of the Sulfolobales order, the majority of species possessing lipoylation systems exclusively retain LplA, or either LipB or LipM, consistent with archaeal genome streamlining. Third, obligate anaerobic archaea display widespread loss of lipoylation and OADHC genes. Conversely, a high level of correspondence is observed between aerobiosis and the presence of LplA/LipB/LipM, LipA and OADHC E2, consistent with the role of lipoylation in aerobic metabolism. This correspondence between OADHC lipoylation capacity and aerobiosis indicates that genomic pathway profiling in archaea is informative and that well characterized pathways may be predictive in relation to abiotic conditions in difficult-to-study extremophiles. Given the highly variable retention of gene repertoires across the archaea, the extension of comparative genomic pathway profiling to broader metabolic and homeostasis networks should be useful in revealing characteristics from metagenomic datasets related to adaptations to diverse environments.     

The Streptomyces coelicolor Lipoate-protein Ligase Is a Circularly Permuted Version of the Escherichia coli Enzyme Composed of Discrete Interacting Domains

Lipoate-protein ligases are used to scavenge lipoic acid from the environment and attach the coenzyme to its cognate proteins, which are generally the E2 components of the 2-oxoacid dehydrogenases. The enzymes use ATP to activate lipoate to its adenylate, lipoyl-AMP, which remains tightly bound in the active site. This mixed anhydride is attacked by the ϵ-amino group of a specific lysine present on a highly conserved acceptor protein domain, resulting in the amide-linked coenzyme. The Streptomyces coelicolor genome encodes only a single putative lipoate ligase. However, this protein had only low sequence identity (<25%) to the lipoate ligases of demonstrated activity and appears to be a circularly permuted version of the known lipoate ligase proteins in that the canonical C-terminal domain seems to have been transposed to the N terminus. We tested the activity of this protein both by in vivo complementation of an Escherichia coli ligase-deficient strain and by in vitro assays. Moreover, when the domains were rearranged into a protein that mimicked the arrangement found in the canonical lipoate ligases, the enzyme retained complementation activity. Finally, when the two domains were separated into two proteins, both domain-containing proteins were required for complementation and catalysis of the overall ligase reaction in vitro. However, only the large domain-containing protein was required for transfer of lipoate from the lipoyl-AMP intermediate to the acceptor proteins, whereas both domain-containing proteins were required to form lipoyl-AMP.     

A novel amidotransferase required for lipoic acid cofactor assembly in Bacillus subtilis

In the companion paper we reported that Bacillus subtilis requires three proteins for lipoic acid metabolism, all of which are members of the lipoate protein ligase family. Two of the proteins, LipM and LplJ, have been shown to be an octanoyltransferase and a lipoate : protein ligase respectively. The third protein, LipL, is essential for lipoic acid synthesis, but had no detectable octanoyltransferase or ligase activity either in vitro or in vivo. We report that LipM specifically modifies the glycine cleavage system protein, GcvH, and therefore another mechanism must exist for modification of other lipoic acid requiring enzymes (e.g. pyruvate dehydrogenase). We show that this function is provided by LipL, which catalyses the amidotransfer (transamidation) of the octanoyl moiety from octanoyl‐GcvH to the E2 subunit of pyruvate dehydrogenase. LipL activity was demonstrated in vitro with purified components and proceeds via a thioester‐linked acyl‐enzyme intermediate. As predicted, ΔgcvH strains are lipoate auxotrophs. LipL represents a new enzyme activity. It is a GcvH:[lipoyl domain] amidotransferase that probably uses a Cys‐Lys catalytic dyad. Although the active site cysteine residues of LipL and LipB are located in different positions within the polypeptide chains, alignment of their structures show these residues occupy similar positions. Thus, these two homologous enzymes have convergent architectures.     

Evidence for two protein-lipoylation activities in Escherichia coli.

The lipoate acyltransferase subunits of the 2-oxo acid dehydrogenase complexes are post-translationally modified with one or more covalently-bound lipoyl cofactors. Two distinct lipoate-protein ligase activities, LPL-A and LPL-B, have been detected in E. coli by their ability to modify purified lipoyl apo-domains of the bacterial pyruvate dehydrogenase complex. Both enzymes require ATP and Mg2+, use L-lipoate, 8-methyllipoate, lipoyl adenylate and octanoyl adenylate as substrates, and both activate lipoyl-deficient pyruvate dehydrogenase complexes. In contrast, only LPL-B uses D-lipoate and octanoate and there are differences in the metal-ion and phosphate requirements. It is suggested that LPL-B may be responsible for the octanoylation of lipoyl domains observed previously under lipoate-deficient conditions.     

Stability of a liposomal formulation containing lipoyl or dihydrolipoyl acylglycerides

Context: The acylglycerides of lipoic and dihydrolipoic acids may serve as slow-release sources for cutaneous delivery of these antioxidants when formulated in a liposomal vehicle.

Objective: Testing was conducted to determine the storage stability of lipoyl glycerides in phospholipid-based liposomes.

Materials and methods: Lipoyl glycerides prepared by transesterification of lipoic acid with high oleic sunflower oil were incorporated into unilamellar liposomes comprised of soy phosphatidylcholine (soyPC) or dioleoylphosphatidylcholine (DOPC).

Results: Lipoyl glycerides were stable in soyPC at 4?°C (90% remaining after five weeks) and decayed with a half-life (t_½) of 14?d at 40?°C. In contrast, lipoyl glycerides embedded in DOPC were completely stable for four weeks at 40?°C. Dihydrolipoyl glycerides in soyPC converted to lipoyl glycerides at 4?°C (t_½?=?14?d) over four weeks, and much more rapidly so at 40?°C (t_½?=?1?d). A hydroperoxide accumulation analysis indicated that lipoyl glycerides and dihydrolipoyl glycerides were modified or degraded while suppressing autoxidation of the polyunsaturated fatty acids present in soyPC. Dynamic light scattering measurements found that liposomes containing lipoyl glycerides or dihydrolipoyl glycerides did not undergo significant size changes for at least 48?d, indicating that inclusion of the lipoic acid derivatives did not induce vesicle aggregation.

Discussion/Conclusion: Substitution of the soyPC with DOPC, which is not readily subject to autoxidation, provided a much more stable storage environment for lipoyl glycerides. These findings confirm the expectation that phospholipid liposomes need to be oxidatively stable vehicles for dermal delivery of lipoic acid derivatives.     

Lipoic acid content of Escherichia coli and other microorganisms

A mutant strain of Escherichia coli K12 requiring lipoic acid, W1485 lip 2 (ATCC 25645), was used to develop a turbidimetric assay for lipoic acid and a polarographic assay based on the oxidation of pyruvate by suspensions of lipoic acid-deficient organisms. The turbidimetric assay was more sensitive with a working range equivalent to 0.2–2.0 ng of dl-α-lipoic acid compared with 5–50 ng for the polarographic method. The mutant responded equally to racemic mixtures of α-lipoic acid, β-lipoic acid and dihydrolipoic acid but gave little response to lipoamide, and other derivatives without prior hydrolysis; 8-methyllipoic acid was a competitive inhibitor of the response to lipoic acid. A high specificity of the mutant for the natural stereoisomer was indicated by the fact that (+)-α-lipoic acid had twice the activity of the racemic mixture. Escherichia coli K12 contained less than 0.05 ng of free (+)-α-lipoic acid per mg dry weight but, depending on the growth substrate, the equivalent of between 13 and 47 ng of (+)-α-lipoic acid per mg dry weight after acid extraction. There was a strong correlation between the lipoic acid content and the sum of the specific activities for the pyruvate and α-ketoglutarate dehydrogenase complexes. Experiments with washed suspensions of Escherichia coli showed only small increases in lipoic acid content (18%) when incubated with pyruvate, cysteine and methionine. When supplied with exogenous lipoic acid the mutant, W1485 lip 2, accumulated very little more than was demanded by its metabolism. The lipoic acid contents of several organisms were measured and correlated with their metabolism.     

Unravelling the lipoyl‐relay of exogenous lipoate utilization in Bacillus subtilis

Lipoate is an essential cofactor for key enzymes of oxidative and one‐carbon metabolism. It is covalently attached to E2 subunits of dehydrogenase complexes and GcvH, the H subunit of the glycine cleavage system. Bacillus subtilis possess two protein lipoylation pathways: biosynthesis and scavenging. The former requires octanoylation of GcvH, insertion of sulfur atoms and amidotransfer of the lipoate to E2s, catalyzed by LipL. Lipoate scavenging is mediated by a lipoyl protein ligase (LplJ) that catalyzes a classical two‐step ATP‐dependent reaction. Although these pathways were thought to be redundant, a ?lipL mutant, in which the endogenous lipoylation pathway of E2 subunits is blocked, showed growth defects in minimal media even when supplemented with lipoate and despite the presence of a functional LplJ. In this study, we demonstrate that LipL is essential to modify E2 subunits of branched chain ketoacid and pyruvate dehydrogenases during lipoate scavenging. The crucial role of LipL during lipoate utilization relies on the strict substrate specificity of LplJ, determined by charge complementarity between the ligase and the lipoylable subunits. This new lipoyl‐relay required for lipoate scavenging highlights the relevance of the amidotransferase as a valid target for the design of new antimicrobial agents among Gram‐positive pathogens.     

Lipoate protein ligase B primarily recognizes the C8-phosphopantetheine arm of its donor substrate and weakly binds the acyl carrier protein

Lipoic acid is a sulfur-containing cofactor indispensable for the function of several metabolic enzymes. In microorganisms, lipoic acid can be salvaged from the surroundings by lipoate protein ligase A (LplA), an ATP-dependent enzyme. Alternatively, it can be synthesized by the sequential actions of lipoate protein ligase B (LipB) and lipoyl synthase (LipA). LipB takes up the octanoyl chain from C₈-acyl carrier protein (C₈-ACP), a byproduct of the type II fatty acid synthesis pathway, and transfers it to a conserved lysine of the lipoyl domain of a dehydrogenase. However, the molecular basis of its substrate recognition is still not fully understood. Using Escherichia coli LipB as a model enzyme, we show here that the octanoyl-transferase mainly recognizes the 4′-phosphopantetheine-tethered acyl-chain of its donor substrate and weakly binds the apo-acyl carrier protein. We demonstrate LipB can accept octanoate from its own ACP and noncognate ACPs, as well as C₈-CoA. Furthermore, our ¹H saturation transfer difference and ³¹P NMR studies demonstrate the binding of adenosine, as well as the phosphopantetheine arm of CoA to LipB, akin to binding to LplA. Finally, we show a conserved ⁷¹RGG⁷³ loop, analogous to the lipoate-binding loop of LplA, is required for full LipB activity. Collectively, our studies highlight commonalities between LipB and LplA in their mechanism of substrate recognition. This knowledge could be of significance in the treatment of mitochondrial fatty acid synthesis related disorders.