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Although a complete pathway of lipoic acid metabolism has been established in Escherichia coli, lipoic acid metabolism in other bacteria is more complex and incompletely understood. Listeria monocytogenes has been shown to utilize two lipoate-protein ligases for lipoic acid scavenging, whereas only one of the ligases can function in utilization of host-derived lipoic acid-modified peptides. We report that lipoic acid scavenging requires not only ligation of lipoic acid but also a lipoyl relay pathway in which an amidotransferase transfers lipoyl groups to the enzyme complexes that require the cofactor for activity. In addition, we provide evidence for a new lipoamidase activity that could allow utilization of lipoyl peptides by lipoate-protein ligase. These data support a model of an expanded, three-enzyme pathway for lipoic acid scavenging that seems widespread in the Firmicutes phylum of bacteria. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Bacillus subtilis lacks a recognizable homologue of the LipB octanoyltransferase, an enzyme essential for lipoic acid synthesis in Escherichia coli LipB transfers the octanoyl moiety from octanoyl-acyl carrier protein to the lipoyl domains of the 2-oxoacid dehydrogenases via a thioester-linked octanoyl-LipH intermediate. The octanoylated dehydrogenase is then converted to the enzyniatically active lipoylated species by insertion of two sulfur atoms into the octanoyl moiety by the S-adenosyl-L-methionine radical enzyme, LipA (lipoate synthase). B. subtilis synthesizes lipoic acid and contains a LipA homologue that is fully functional in E. coli. Therefore, the lack of a LipB homologue presented the puzzle of how B. subtilis synthesizes the LipA substrate. We report that B. subtilis encodes an octanoyltransferase that has virtually no sequence resemblance to E. coli LipB but instead has a sequence that resembles that of the E. coli lipoate ligase. LplA. On the basis of this resemblance, these genes have generally been annotated as encoding a lipoate ligase, an enzyme that in E. coli scavenges lipoic acid from the environment but plays no role tn de novo synthesis. We have named the B. stsbtilis octanoyltransferase LipM and find that, like LipB, the LipM reaction proceeds through a thioester-linked acyl enzyme intermediate. The LipM active Site nucleophile was identified as Cl 50 by the finding that this thiol becomes modified when LipM is expressed in E. coli. The level of the octanoyl- LipM intermediate can be significantly decreased by blocking fatty acid synthesis during LipM expression, and Cl 50 was confirmed as an essential active site residue by site-directed mutagenesis. LipM homologues seem the sole type of octanoyltransferase present in the firmicittes and are also present in the cyanobacteria. LipM type octanoyltransferases represent a new dade of the PF03099 protein family, suggesting that octanoyl transfer activity has evolved at least twice within this superfamily. [ABSTRACT FROM AUTHOR]

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 eamino 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 Lp1A family detected in silico have only the large catalytic domain. Two x-ray structures of the Thermoplasma acidophilum LpIA 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, LpIB. We found that complementation of Escherichia coli strains lacking lipoate ligase with T, acidophilum LpIA was possible only when LplB was also present. LpIA had no detectable ligase activity in vitro in the absence of LpIB. Moreover LplA and LpIB 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. [ABSTRACT FROM AUTHOR]