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1.
The Escherichia coli lipB gene encodes lipoyl (octanoyl)-acyl carrier protein:protein transferase
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In an earlier study (S. W. Jordan and J. E. Cronan, Jr., J. Biol. Chem. 272:17903-17906, 1997) we reported a new enzyme, lipoyl-[acyl carrier protein]-protein N-lipoyltransferase, in Escherichia coli and mitochondria that transfers lipoic acid from lipoyl-acyl carrier protein to the lipoyl domains of pyruvate dehydrogenase. It was also shown that E. coli lipB mutants lack this enzyme activity, a finding consistent with lipB being the gene that encoded the lipoyltransferase. However, it remained possible that lipB encoded a positive regulator required for lipoyltransferase expression or action. We now report genetic and biochemical evidence demonstrating that lipB encodes the lipoyltransferase. A lipB temperature-sensitive mutant was shown to produce a thermolabile lipoyltransferase and a tagged version of the lipB-encoded protein was purified to homogeneity and shown to catalyze the transfer of either lipoic acid or octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. In the course of these experiments the ATG initiation codon commonly assigned to lipB genes in genomic databases was shown to produce a nonfunctional E. coli LipB protein, whereas initiation at an upstream TTG codon gave a stable and enzymatically active protein. Prior genetic results (T. W. Morris, K. E. Reed, and J. E. Cronan, Jr., J. Bacteriol. 177:1-10, 1995) suggested that lipoate protein ligase (LplA) could also utilize (albeit poorly) acyl carrier protein substrates in addition to its normal substrates lipoic acid plus ATP. We have detected a very slow LplA-catalyzed transfer of lipoic acid and octanoic acid from their acyl carrier protein thioesters to the lipoyl domain of pyruvate dehydrogenase. A nonhydrolyzable lipoyl-AMP analogue was found to competitively inhibit both ACP-dependent and ATP-dependent reactions of LplA, suggesting that the same active site catalyzes two chemically diverse reactions. 相似文献
2.
Miller JR Busby RW Jordan SW Cheek J Henshaw TF Ashley GW Broderick JB Cronan JE Marletta MA 《Biochemistry》2000,39(49):15166-15178
The Escherichia coli lipA gene product has been genetically linked to carbon-sulfur bond formation in lipoic acid biosynthesis [Vanden Boom, T. J., Reed, K. E., and Cronan, J. E., Jr. (1991) J. Bacteriol. 173, 6411-6420], although in vitro lipoate biosynthesis with LipA has never been observed. In this study, the lipA gene and a hexahistidine tagged lipA construct (LipA-His) were overexpressed in E. coli as soluble proteins. The proteins were purified as a mixture of monomeric and dimeric species that contain approximately four iron atoms per LipA polypeptide and a similar amount of acid-labile sulfide. Electron paramagnetic resonance and electronic absorbance spectroscopy indicate that the proteins contain a mixture of [3Fe-4S] and [4Fe-4S] cluster states. Reduction with sodium dithionite results in small quantities of an S = 1/2 [4Fe-4S](1+) cluster with the majority of the protein containing a species consistent with an S = 0 [4Fe-4S](2+) cluster. LipA was assayed for lipoate or lipoyl-ACP formation using E. coli lipoate-protein ligase A (LplA) or lipoyl-[acyl-carrier-protein]-protein-N-lipoyltransferase (LipB), respectively, to lipoylate apo-pyruvate dehydrogenase complex (apo-PDC) [Jordan, S. W., and Cronan, J. E. (1997) Methods Enzymol. 279, 176-183]. When sodium dithionite-reduced LipA was incubated with octanoyl-ACP, LipB, apo-PDC, and S-adenosyl methionine (AdoMet), lipoylated PDC was formed. As shown by this assay, octanoic acid is not a substrate for LipA. Confirmation that LipA catalyzes formation of lipoyl groups from octanoyl-ACP was obtained by MALDI mass spectrometry of a recombinant PDC lipoyl-binding domain that had been lipoylated in a LipA reaction. These results provide information about the mechanism of LipA catalysis and place LipA within the family of iron-sulfur proteins that utilize AdoMet for radical-based chemistry. 相似文献
3.
4.
Chromosomal amplification of the Escherichia coli lipB region confers high-level resistance to selenolipoic acid
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One of the mutants (slr7 mutant) of a wild-type Escherichia coli strain resistant to selenolipoic acid reported previously (K. E. Reed, T. W. Morris, and J. E. Cronan, Jr., Proc. Natl. Acad. Sci. USA 91:3720-3724, 1994) unexpectedly grew on minimal medium following transductional introduction of a lipA null mutation. We report that the slr7 strain carries a duplication of the lip chromosomal region that causes the phenotype of the mutant strain. 相似文献
5.
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. 相似文献
6.
Lipoyl-lysine swinging arms are crucial to the reactions catalysed by the 2-oxo acid dehydrogenase multienzyme complexes. A gene encoding a putative lipoate protein ligase (LplA) of Thermoplasma acidophilum was cloned and expressed in Escherichia coli. The recombinant protein, a monomer of molecular mass 29 kDa, was catalytically inactive. Crystal structures in the absence and presence of bound lipoic acid were solved at 2.1 A resolution. The protein was found to fall into the alpha/beta class and to be structurally homologous to the catalytic domains of class II aminoacyl-tRNA synthases and biotin protein ligase, BirA. Lipoic acid in LplA was bound in the same position as biotin in BirA. The structure of the T.acidophilum LplA and limited proteolysis of E.coli LplA together highlighted some key features of the post-translational modification. A loop comprising residues 71-79 in the T.acidophilum ligase is proposed as interacting with the dithiolane ring of lipoic acid and discriminating against the entry of biotin. A second loop comprising residues 179-193 was disordered in the T.acidophilum structure; tryptic cleavage of the corresponding loop in the E.coli LplA under non-denaturing conditions rendered the enzyme catalytically inactive, emphasizing its importance. The putative LplA of T.acidophilum lacks a C-terminal domain found in its counterparts in E.coli (Gram-negative) or Streptococcus pneumoniae (Gram-positive). A gene encoding a protein that appears to have structural homology to the additional domain in the E.coli and S.pneumoniae enzymes was detected alongside the structural gene encoding the putative LplA in the T.acidophilum genome. It is likely that this protein is required to confer activity on the LplA as currently purified, one protein perhaps catalysing the formation of the obligatory lipoyl-AMP intermediate, and the other transferring the lipoyl group from it to the specific lysine residue in the target protein. 相似文献
7.
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. 相似文献
8.
Global Conformational Change Associated with the Two-step Reaction Catalyzed by Escherichia coli Lipoate-Protein Ligase A 总被引:1,自引:0,他引:1
Kazuko Fujiwara Nobuo Maita Harumi Hosaka Kazuko Okamura-Ikeda Atsushi Nakagawa Hisaaki Taniguchi 《The Journal of biological chemistry》2010,285(13):9971-9980
Lipoate-protein ligase A (LplA) catalyzes the attachment of lipoic acid to lipoate-dependent enzymes by a two-step reaction: first the lipoate adenylation reaction and, second, the lipoate transfer reaction. We previously determined the crystal structure of Escherichia coli LplA in its unliganded form and a binary complex with lipoic acid (Fujiwara, K., Toma, S., Okamura-Ikeda, K., Motokawa, Y., Nakagawa, A., and Taniguchi, H. (2005) J Biol. Chem. 280, 33645–33651). Here, we report two new LplA structures, LplA·lipoyl-5′-AMP and LplA·octyl-5′-AMP·apoH-protein complexes, which represent the post-lipoate adenylation intermediate state and the pre-lipoate transfer intermediate state, respectively. These structures demonstrate three large scale conformational changes upon completion of the lipoate adenylation reaction: movements of the adenylate-binding and lipoate-binding loops to maintain the lipoyl-5′-AMP reaction intermediate and rotation of the C-terminal domain by about 180°. These changes are prerequisites for LplA to accommodate apoprotein for the second reaction. The Lys133 residue plays essential roles in both lipoate adenylation and lipoate transfer reactions. Based on structural and kinetic data, we propose a reaction mechanism driven by conformational changes. 相似文献
9.
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." 相似文献
10.
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-LipB intermediate. The octanoylated dehydrogenase is then converted to the enzymatically 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 in de novo synthesis. We have named the B. subtilis 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 C150 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 C150 was confirmed as an essential active site residue by site-directed mutagenesis. LipM homologues seem the sole type of octanoyltransferase present in the firmicutes and are also present in the cyanobacteria. LipM type octanoyltransferases represent a new clade of the PF03099 protein family, suggesting that octanoyl transfer activity has evolved at least twice within this superfamily. 相似文献
11.
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. 相似文献
12.
Structure and selectivity in post-translational modification: attaching the biotinyl-lysine and lipoyl-lysine swinging arms in multifunctional enzymes.
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The post-translational attachment of biotin and lipoic acid to specific lysine residues displayed in protruding beta-turns in homologous biotinyl and lipoyl domains of their parent enzymes is catalysed by two different ligases. We have expressed in Escherichia coli a sub-gene encoding the biotinyl domain of E.coli acetyl-CoA carboxylase, and by a series of mutations converted the protein from the target for biotinylation to one for lipoylation, in vivo and in vitro. The biotinylating enzyme, biotinyl protein ligase (BPL), and the lipoylating enzyme, LplA, exhibited major differences in the recognition process. LplA accepted the highly conserved MKM motif that houses the target lysine residue in the biotinyl domain beta-turn, but was responsive to structural cues in the flanking beta-strands. BPL was much less sensitive to changes in these beta-strands, but could not biotinylate a lysine residue placed in the DKA motif characteristic of the lipoyl domain beta-turn. The presence of a further protruding thumb between the beta2 and beta3 strands in the wild-type biotinyl domain, which has no counterpart in the lipoyl domain, is sufficient to prevent aberrant lipoylation in E.coli. The structural basis of this discrimination contrasts with other forms of post-translational modification, where the sequence motif surrounding the target residue can be the principal determinant. 相似文献
13.
Kim DJ Kim KH Lee HH Lee SJ Ha JY Yoon HJ Suh SW 《The Journal of biological chemistry》2005,280(45):38081-38089
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. 相似文献
14.
Fujiwara K Toma S Okamura-Ikeda K Motokawa Y Nakagawa A Taniguchi H 《The Journal of biological chemistry》2005,280(39):33645-33651
Lipoate-protein ligase A (LplA) catalyzes the formation of lipoyl-AMP from lipoate and ATP and then transfers the lipoyl moiety to a specific lysine residue on the acyltransferase subunit of alpha-ketoacid dehydrogenase complexes and on H-protein of the glycine cleavage system. The lypoyllysine arm plays a pivotal role in the complexes by shuttling the reaction intermediate and reducing equivalents between the active sites of the components of the complexes. We have determined the X-ray crystal structures of Escherichia coli LplA alone and in a complex with lipoic acid at 2.4 and 2.9 angstroms resolution, respectively. The structure of LplA consists of a large N-terminal domain and a small C-terminal domain. The structure identifies the substrate binding pocket at the interface between the two domains. Lipoic acid is bound in a hydrophobic cavity in the N-terminal domain through hydrophobic interactions and a weak hydrogen bond between carboxyl group of lipoic acid and the Ser-72 or Arg-140 residue of LplA. No large conformational change was observed in the main chain structure upon the binding of lipoic acid. 相似文献
15.
Lipoate is an essential cofactor for key enzymes of oxidative metabolism. Plasmodium falciparum possesses genes for lipoate biosynthesis and scavenging, but it is not known if these pathways are functional, nor what their relative contribution to the survival of intraerythrocytic parasites might be. We detected in parasite extracts four lipoylated proteins, one of which cross-reacted with antibodies against the E2 subunit of apicoplast-localized pyruvate dehydrogenase (PDH). Two highly divergent parasite lipoate ligase A homologues (LplA), LipL1 (previously identified as LplA) and LipL2, restored lipoate scavenging in lipoylation-deficient bacteria, indicating that Plasmodium has functional lipoate-scavenging enzymes. Accordingly, intraerythrocytic parasites scavenged radiolabelled lipoate and incorporated it into three proteins likely to be mitochondrial. Scavenged lipoate was not attached to the PDH E2 subunit, implying that lipoate scavenging drives mitochondrial lipoylation, while apicoplast lipoylation relies on biosynthesis. The lipoate analogue 8-bromo-octanoate inhibited LipL1 activity and arrested P. falciparum in vitro growth, decreasing the incorporation of radiolabelled lipoate into parasite proteins. Furthermore, growth inhibition was prevented by lipoate addition in the medium. These results are consistent with 8-bromo-octanoate specifically interfering with lipoate scavenging. Our study suggests that lipoate metabolic pathways are not redundant, and that lipoate scavenging is critical for Plasmodium intraerythrocytic survival. 相似文献
16.
Natalí B. Rasetto Antonela Lavatelli Natalia Martin María Cecilia Mansilla 《Molecular microbiology》2019,112(1):302-316
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. 相似文献
17.
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 Km 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. 相似文献
18.
Typhanie Bouvenot Amlie Dewitte Nadia Bennaceur Elizabeth Pradel Franois Pierre Sbastien Bontemps-Gallo Florent Sebbane 《The ISME journal》2021,15(4):1136
To thrive, vector-borne pathogens must survive in the vector’s gut. How these pathogens successfully exploit this environment in time and space has not been extensively characterized. Using Yersinia pestis (the plague bacillus) and its flea vector, we developed a bioluminescence-based approach and employed it to investigate the mechanisms of pathogenesis at an unprecedented level of detail. Remarkably, lipoylation of metabolic enzymes, via the biosynthesis and salvage of lipoate, increases the Y. pestis transmission rate by fleas. Interestingly, the salvage pathway’s lipoate/octanoate ligase LplA enhances the first step in lipoate biosynthesis during foregut colonization but not during midgut colonization. Lastly, Y. pestis primarily uses lipoate provided by digestive proteolysis (presumably as lipoyl peptides) rather than free lipoate in blood, which is quickly depleted by the vector. Thus, spatial and temporal factors dictate the bacterium’s lipoylation strategies during an infection, and replenishment of lipoate by digestive proteolysis in the vector might constitute an Achilles’ heel that is exploited by pathogens.Subject terms: Bacterial pathogenesis, Metabolism 相似文献
19.
Martin N Christensen QH Mansilla MC Cronan JE de Mendoza D 《Molecular microbiology》2011,80(2):335-349
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. 相似文献
20.
Apicomplexan parasites contain a vestigial plastid called apicoplast which has been suggested to be a site of [Fe-S] cluster biogenesis. Here we report the cloning of lipoic acid synthase (LipA) from Toxoplasma gondii, a well known [Fe-S] protein. It is able to complement a LipA-deficient Escherichia coli strain, clearly demonstrating that the parasite protein is a functional LipA. The N-terminus of T. gondii LipA is unusual with respect to an internal signal peptide preceding an apicoplast targeting domain. Nevertheless, it efficiently targets a reporter protein to the apicoplast of T. gondii whereas co-localization with the fluorescently labeled mitochondrion was not detected. In silico analysis of several apicomplexan genomes indicates that the parasites, in addition to the presumably apicoplast-resident pyruvate dehydrogenase complex, contain three other mitochondrion-localized target proteins for lipoic acid attachment. We also identified single genes for lipoyl (octanoyl)-acyl carrier protein:protein transferase (LipB) and lipoate protein ligase (LplA) in these genomes. It thus appears that unlike plants, which have only two LipA and LipB isoenzymes in both the chloroplasts and the mitochondria, Apicomplexa seem to use the second known lipoylating activity, LplA, for lipoylation in their mitochondrion. 相似文献