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.     

Lipoylating and biotinylating enzymes contain a homologous catalytic module

Biotin and lipoic acid moieties are the covalently attached coenzyme cofactors of several multicomponent enzyme complexes that catalyze key metabolic reactions. Attachment of these moieties to the biotinyl- and lipoyl-dependent enzymes is post-translationally catalyzed by specific biotinylating and lipoylating protein enzymes. In Escherichia coli, two different enzymes, LplA and LipB, catalyze independent pathways for the lipoylation of the relevant enzymes, whereas only one enzyme, the BirA protein, is responsible for all the biotinylation. Counterparts of the E. coli BirA, LplA, and LipB enzymes have been previously identified in many organisms, but homology among the three families has never been reported. Computational analysis based on PSI-BLAST profiles and secondary structure predictions indicates, however, that lipoylating and biotinylating enzymes are evolutionarily related protein families containing a homologous catalytic module. Sequence conservation among the three families is very poor, but a single lysine residue is strictly conserved in all of them, which, according to the available X-ray crystal structure of the E. coli BirA protein, is expected to contribute to the binding of lipoic acid in the LplA and LipB enzymes.     

Structure-guided engineering of a Pacific Blue fluorophore ligase for specific protein imaging in living cells

Mutation of a gatekeeper residue, tryptophan 37, in E. coli lipoic acid ligase (LplA), expands substrate specificity such that unnatural probes much larger than lipoic acid can be recognized. This approach, however, has not been successful for anionic substrates. An example is the blue fluorophore Pacific Blue, which is isosteric to 7-hydroxycoumarin and yet not recognized by the latter's ligase ((W37V)LplA) or any tryptophan 37 point mutant. Here we report the results of a structure-guided, two-residue screening matrix to discover an LplA double mutant, (E20G/W37T)LplA, that ligates Pacific Blue as efficiently as (W37V)LplA ligates 7-hydroxycoumarin. The utility of this Pacific Blue ligase for specific labeling of recombinant proteins inside living cells, on the cell surface, and inside acidic endosomes is demonstrated.     

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."     

The Escherichia coli lipB gene encodes lipoyl (octanoyl)-acyl carrier protein:protein transferase

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.     

Structure and selectivity in post-translational modification: attaching the biotinyl-lysine and lipoyl-lysine swinging arms in multifunctional enzymes.

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.     

Expression and purification of E. coli BirA biotin ligase for in vitro biotinylation

The extremely tight binding between biotin and avidin or streptavidin makes labeling proteins with biotin a useful tool for many applications. BirA is the Escherichia coli biotin ligase that site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (also known as Avi-tag). As a complementary approach to in vivo biotinylation of Avi-tag-bearing proteins, we developed a protocol for producing recombinant BirA ligase for in vitro biotinylation. The target protein was expressed as both thioredoxin and MBP fusions, and was released from the corresponding fusion by TEV protease. The liberated ligase was separated from its carrier using HisTrap HP column. We obtained 24.7 and 27.6 mg BirA ligase per liter of culture from thioredoxin and MBP fusion constructs, respectively. The recombinant enzyme was shown to be highly active in catalyzing in vitro biotinylation. The described protocol provides an effective means for making BirA ligase that can be used for biotinylation of different Avi-tag-bearing substrates.     

Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes

Live cell imaging is a powerful method to study protein dynamics at the cell surface, but conventional imaging probes are bulky, or interfere with protein function, or dissociate from proteins after internalization. Here, we report technology for covalent, specific tagging of cellular proteins with chemical probes. Through rational design, we redirected a microbial lipoic acid ligase (LplA) to specifically attach an alkyl azide onto an engineered LplA acceptor peptide (LAP). The alkyl azide was then selectively derivatized with cyclo-octyne conjugates to various probes. We labeled LAP fusion proteins expressed in living mammalian cells with Cy3, Alexa Fluor 568 and biotin. We also combined LplA labeling with our previous biotin ligase labeling, to simultaneously image the dynamics of two different receptors, coexpressed in the same cell. Our methodology should provide general access to biochemical and imaging studies of cell surface proteins, using small fluorophores introduced via a short peptide tag.     

Lipoic acid synthesis: a new family of octanoyltransferases generally annotated as lipoate protein ligases

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.     

Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon.

The Bacillus subtilis birA gene, which regulates biotin biosynthesis, has been cloned and characterized. The birA gene maps at 202 degrees on the B. subtilis chromosome and encodes a 36,200-Da protein that is 27% identical to Escherichia coli BirA protein. Three independent mutations in birA that lead to deregulation of biotin synthesis alter single amino acids in the amino-terminal end of the protein. The amino-terminal region that is affected by these three birA mutations shows sequence similarity to the helix-turn-helix DNA binding motif previously identified in E. coli BirA protein. B. subtilis BirA protein also possesses biotin-protein ligase activity, as judged by its ability to complement a conditional lethal birA mutant of E. coli.     

The C-terminal domain of biotin protein ligase from E. coli is required for catalytic activity

Biotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl-5'-AMP, the catalytic intermediate in the biotinylation reaction. We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA. Here we used site-directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP. None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild-type levels when assayed with our BCCP mutant substrates. However, a BirA variant, in which K277 of the C-terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild-type BirA. No function has been identified previously for the BirA C-terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site. Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C-terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25-fold decrease in the affinity for ATP. Our data indicate that the C-terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain.     

Crystal structure of lipoate-protein ligase A from Escherichia coli. Determination of the lipoic acid-binding site

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.     

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.     

Novel system for in vivo biotinylation and its application to crab antimicrobial protein scygonadin

BirA is a biotin ligase from Escherichia coli that specifically biotinylates a lysine side-chain within a 15-amino acid acceptor peptide (also known as Avi-tag). We developed a protocol for producing recombinant BirA ligase in E. coli for in vitro biotinylation (Li and Sousa, Prot Expr Purif, 82:162-167, 2012) in which the target protein was expressed as both thioredoxin and MBP fusions, and was released by TEV protease-mediated cleavage. The liberated ligase and the fusion proteins were enzymatically active. Based on that observation, we have now developed a novel system for in vivo biotinylation by co-expressing the Avi-tagged target protein with the MBP-BirA fusion. The effectiveness of this system was demonstrated by the successful in vivo labeling of antimicrobial protein, scygonadin. This new system shows improved efficiency compared with pre-existing one and this is likely attributed to the high expression level and solubility of the co-expressed MBP-BirA.     

Apicomplexan parasites contain a single lipoic acid synthase located in the plastid

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.     

Co-repressor induced order and biotin repressor dimerization: a case for divergent followed by convergent evolution

BirA catalyzes the adenylation and subsequent covalent attachment of biotin to the biotin carboxyl carrier protein (BCCP). In the absence of apo-BCCP, biotin-5'-AMP acts as a co-repressor that induces BirA dimerization and binding to the bio operator to repress biotin biosynthesis. The crystal structures of apo-BirA, and BirA in complex with biotin have been reported. We here describe the 2.8A resolution crystal structure of BirA in complex with the co-repressor analog biotinol-5'-AMP. It was previously shown that the structure of apo-BirA is monomeric and that binding of biotin weakly induces a dimeric structure in which three disordered surface loops become organized to form the dimer interface. The structure of the co-repressor complex is also a dimer, clearly related to the BirA.biotin structure, but with several significant conformational changes. A hitherto disordered "adenylate binding loop" forms a well-defined structure covering the co-repressor. The co-repressor buttresses the dimer interface, resulting in improved packing and a 12 degrees change in the hinge-bending angle along the dimer interface relative to the BirA.biotin structure. This helps explain why the binding of the co-repressor is necessary to optimize the binding of BirA to the bioO operator. The structure reveals an unexpected use of the nucleotide-binding motif GXGXXG in binding adenylate and controlling the repressor function. Finally, based on structural analysis we propose that the class of adenylating enzymes represented by BirA, lipoate protein ligase and class II tRNA synthetases diverged early and were selected based on their ability to sequester co-factors or amino acid residues, and adenylation activity arose independently through functional convergence.     

Targeted and proximity-dependent promiscuous protein biotinylation by a mutant Escherichia coli biotin protein ligase

A method for general protein biotinylation by enzymatic means has been developed. A mutant form (R118G) of the biotin protein ligase (BirA) of Escherichia coli is used and biotinylation is thought to proceed by chemical acylation of protein lysine side chains by biotinoyl-5'-AMP released from the mutant protein. Bovine serum albumin, chloramphenicol acetyltransferase, immunoglobulin chains and RNAse A as well as a large number of E. coli proteins have been biotinylated. The biotinylation reaction is proximity dependent in that the extent of biotinylation is much greater when the ligase is coupled to the acceptor protein than when the acceptor is free in solution. This is presumably due to rapid hydrolysis of the acylation agent, biotinoyl-5'-AMP. Therefore, when the mutant ligase is attached to one partner involved in a protein-protein interaction, it can be used to specifically tag the other partner with biotin, thereby permitting facile detection and recovery of the proteins by existing avidin/streptavidin technology.     

Development of an enzymatic method for site-specific incorporation of desthiobiotin to recombinant proteins in vitro

To extend the (strept)avidin-biotin technology for affinity purification of proteins, development of reusable biochips and immobilized enzyme bioreactors, selective immobilization of a protein of interest from a crude sample to a protein array without protein purification and many other possible applications, the (strept)avidin-biotin interaction is better when reversible. A gentle enzymatic method to introduce a biotin analog, desthiobiotin, in a site-specific manner to recombinant proteins carrying a biotinylation tag has been developed. The optimal condition for efficient in vitro desthiobiotinylation catalyzed by Escherichia coli biotin ligase (BirA) in 1-4h has been established by systematically varying the substrate concentrations, reaction time, and pH. Real desthiobiotinylation in the absence of any significant biotinylation using this enzymatic method was confirmed by mass spectrometric analysis of the desthiobiotinylated tag. This approach was applied to affinity purify desthiobiotinylated staphylokinase secreted by recombinant Bacillus subtilis to high purity and with good recovery using streptavidin-agarose. The matrix can be regenerated for reuse. This study represents the first successful application of E. coli BirA to incorporate biotin analog to recombinant proteins in a site-specific manner.