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At least 271 records · Page 15Linked to original sources
PubMed1997

Effects of base mismatches on joining of short oligodeoxynucleotides by DNA ligases.

The requirement for Watson-Crick base pairing surrounding a nick in duplex DNA to be sealed by DNA ligase is the basis for oligonucleotide ligation assays that distinguish single base mutations in DNA targets. Experiments in a model system demonstrate that the minimum length of oligonucleotide that can be joined differs for different ligases. Thermus thermophilus (Tth) DNA ligase is unable to join any oligonucleotide of length six or less, while T4 DNA ligase and T7 DNA ligase are both able to join hexamers. The rate of oligonucleotide ligation by Tth DNA ligase increases between heptamer and nonamer. Mismatches which cause the duplex to be shortened by fraying, at the end distal to the join, slow the ligation reaction. In the case of Tth DNA ligase, mismatches at the seventh and eighth position 5'to the nick completely inhibit the ligation of octamers. The results are relevant to mechanisms of ligation.

Bacteriophage T7↗
PubMed1998

Specificity and fidelity of strand joining by Chlorella virus DNA ligase.

Chlorella virus PBCV-1 DNA ligase seals nicked duplex DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging template strand, but cannot ligate a nicked duplex composed of two DNAs annealed on an RNA template. Whereas PBCV-1 ligase efficiently joins a 3'-OH RNA to a 5'-phosphate DNA, it is unable to join a 3'-OH DNA to a 5'-phosphate RNA. The ligase discriminates at the substrate binding step between nicked duplexes containing 5'-phosphate DNA versus 5'-phosphate RNA strands. PBCV-1 ligase readily seals a nicked duplex DNA containing a single ribonucleotide substitution at the reactive 5'-phosphate end. These results suggest a requirement for a B-form helical conformation of the polynucleotide on the 5'-phosphate side of the nick. Single base mismatches at the nick exert disparate effects on DNA ligation efficiency. PBCV-1 ligase tolerates mismatches involving the 5'-phosphate nucleotide, with the exception of 5'-A:G and 5'-G:A mispairs, which reduce ligase activity by two orders of magnitude. Inhibitory configurations at the 3'-OH nucleotide include 3'-G:A, 3'-G:T, 3'-T:T, 3'-A:G, 3'-G:G, 3'-A:C and 3'-C:C. Our findings indicate that Chlorella virus DNA ligase has the potential to affect genome integrity by embedding ribonucleotides in viral DNA and by sealing nicked molecules with mispaired ends, thereby generating missense mutations.

DNA↗
PubMed1999

Ligase-based detection of mononucleotide repeat sequences.

Up to 15% of all colorectal cancers are considered to be replication error positive (RER(+)) and contain mutations at hundreds of thousands of microsatellite repeat sequences. Recently, a number of intragenic mononucleotide repeat sequences have been demonstrated to be targets for inactivating genes in RER(+)colorectal tumors. In this study, thermostable DNA ligases were tested for the ability to detect alterations in microsatellite sequences in colon tumor samples. Ligation profiles on mononucleotide repeat sequences were determined for four related thermostable DNA ligases, Thermus thermophilus ( Tth ) ligase, Thermus sp. AK16D ligase, Aquifex aeolicus ligase and the K294R mutant of the Tth ligase. While the limit of detection for point mutations was one mutation in 1000 wild-type sequences, the ability to detect a single base deletion in a 10 base mononucleotide repeat was one mutation in 100 wild-type sequences. Furthermore, the misligation error increased exponentially as the length of the mono-nucleotide repeat increased, and was 10% of the correct signal for a 19 base mononucleotide repeat. A fluorescent ligase-based assay [polymerase chain reaction/ligase detection reaction (PCR/LDR)] correlated with results obtained using a radioactive assay to detect instability within the TGF-beta Type II receptor gene. PCR/LDR was also used to detect the APCI1307K mononucleotide repeat allele which has a carrier frequency of 6.1% in Ashkenazi Jewish individuals. In a blind study, 30 samples that had been typed for the presence of the APCI1307K allele were tested. The PCR/LDR results correlated with those obtained using sequencing and allele-specific oligonucleotide hybridization for 16 samples carrying the mutation and 13 wild-type samples. Ligation assays that characterize mononucleotide repeats can be used to rapidly detect somatic mutations in tumors, and to screen for individuals who have a hereditary predisposition to develop colon cancer.

Adenomatous Polyposis Coli↗
PubMed2001

A second NAD(+)-dependent DNA ligase (LigB) in Escherichia coli.

Escherichia coli DNA ligase (LigA) is the prototype of the NAD(+)-dependent class of DNA ligases found in all bacteria. Here we report the characterization of E.coli LigB, a second NAD(+)-dependent DNA ligase identified by virtue of its sequence similarity to LigA. LigB differs from LigA in that it lacks the BRCA1 C-terminus domain (BRCT) and two of the four Zn-binding cysteines that are present in LigA and all other bacterial NAD(+) ligases. We found that recombinant LigB catalyzed strand joining on a singly-nicked DNA in the presence of a divalent cation and NAD(+), and that LigB reacted with NAD(+) to form a covalent ligase-adenylate intermediate. Alanine substitution for the motif I lysine ((126)KxDG) abolished nick joining and ligase-adenylate formation by LigB, thus confirming that the ligase and adenylyltransferase activities are intrinsic to the LigB protein.

Alanine↗
PubMed1997

Nick sensing by vaccinia virus DNA ligase requires a 5' phosphate at the nick and occupancy of the adenylate binding site on the enzyme.

Vaccinia virus DNA ligase has an intrinsic nick-sensing function. The enzyme discriminates at the substrate binding step between a DNA containing a 5' phosphate and a DNA containing a 5' hydroxyl at the nick. Further insights into nick recognition and catalysis emerge from studies of the active-site mutant K231A, which is unable to form the covalent ligase-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K231A does catalyze phosphodiester bond formation at a preadenylated nick. Hence, the active-site lysine of DNA ligase is not required for the strand closure step of the ligation reaction. The K231A mutant binds tightly to nicked DNA-adenylate but has low affinity for a standard DNA nick. The wild-type vaccinia virus ligase, which is predominantly ligase-adenylate, binds tightly to a DNA nick. This result suggests that occupancy of the AMP binding pocket of DNA ligase is essential for stable binding to DNA. Sequestration of an extrahelical nucleotide by DNA-bound ligase is reminiscent of the base-flipping mechanism of target-site recognition and catalysis used by other DNA modification and repair enzymes.

Adenosine Monophosphate↗
PubMed2005

Structure-function analysis of yeast tRNA ligase.

Trl 1 is an essential 827-amino-acid enzyme that executes the end-healing and end-sealing steps of tRNA splicing in Saccharomyces cerevisiae. Trl1 consists of two catalytic domains--an N-terminal adenylyltransferase/ligase component (amino acids 1-388) and a C-terminal 5'-kinase/cyclic phosphodiesterase component (amino acids 389-827)--that can function in tRNA splicing in vivo when expressed as separate polypeptides. Sedimentation analysis indicates that the ligase and kinase/CPD domains are monomeric proteins that do not form a stable complex in trans. To understand the structural requirements for the RNA ligase component, we performed a mutational analysis of amino acids that are conserved in Trl1 homologs from other fungi. Alanine scanning identified 23 new residues as essential for Trl1-(1-388) activity in vivo. Structure-activity relationships at these positions, and four essential residues defined previously, were clarified by introducing 50 different conservative substitutions. Lethal mutations of Lys114, Glu184, Glu266, and Lys284 abolished Trl1 adenylyltransferase activity in vitro. The essential elements embrace (1) putative equivalents of nucleotidyltransferase motifs I, Ia, III, IV, and V found in DNA ligases, T4 RNA ligase 2, and mRNA capping enzymes; (2) an N-terminal segment shared with the T4 RNA ligase 1 subfamily only; and (3) a constellation of conserved residues specific to fungal tRNA splicing enzymes. We identify yeastlike tRNA ligases in the proteomes of Leishmania and Trypanosoma. These findings recommend tRNA ligase as a target for antifungal and antiprotozoal drug discovery.

Amino Acid Motifs↗
PubMed2006

Novel RING E3 ubiquitin ligases in breast cancer.

Defects in ubiquitin E3 ligases are implicated in the pathogenesis of several human diseases, including cancer, because of their central role in the control of diverse signaling pathways. RING E3 ligases promote the ubiquitination of proteins that are essential to a variety of cellular events. Identification of which ubiquitin ligases specifically affect distinct cellular processes is essential to the development of targeted therapeutics for these diseases. Here we discuss two novel RING E3 ligases, BCA2 and RNF11, that are closely linked to human breast cancer. BCA2 E3 ligase is coregulated with estrogen receptor and plays a role in the regulation of epidermal growth factor receptor (EGF-R) trafficking. RNF11 is a small RING E3 ligase that affects transforming growth factorbeta and EGF-R signaling and is overexpressed in invasive breast cancers. These two proteins demonstrate the complexity of RING E3 ligase interactions in breast cancer and are potential targets for therapeutic interventions.

Breast Neoplasms↗
PubMed1999

Rejoining kinetics of DNA single- and double-strand breaks in normal and DNA ligase-deficient cells after exposure to ultraviolet C and gamma radiation: an evaluation of ligating activities involved in different DNA repair processes.

The repair kinetics for rejoining of DNA single- and double-strand breaks after exposure to UVC or gamma radiation was measured in cells with deficiencies in DNA ligase activities and in their normal counterparts. Human 46BR cells were deficient in DNA ligase I. Hamster EM9 and EM-C11 cells were deficient in DNA ligase III activity as a consequence of mutations in the XRCC1 gene. Hamster XR-1 cells had mutation in the XRCC4 gene, whose product stimulates DNA ligase IV activity. DNA single- and double-strand breaks were assessed by the comet assay in alkaline conditions and by the technique of graded-field gel electrophoresis in neutral conditions, respectively. 46BR cells, which are known to re-ligate at a reduced rate the DNA single-strand breaks incurred during processing of damage induced by UVC but not gamma radiation, were shown to have a normal repair of radiation-induced DNA double-strand breaks. EM9 cells exhibited a reduced rate of rejoining of DNA single-strand breaks after exposure to ionizing radiation, as reported previously, as well as UVC radiation. EM-C11 cells were deficient in the repair of radiation-induced-DNA single-strand breaks but, in contrast to EM9 cells, demonstrated the same kinetics as the parental cell line in the resealing of DNA breaks resulting from exposure to UVC radiation. Both EM9 and EM-C11 cells displayed a significant defect in rejoining of radiation-induced-DNA double-strand breaks. XR-1 cells were confirmed to be highly deficient in the repair of radiation-induced DNA double-strand breaks but appeared to rejoin DNA single-strand breaks after UVC and gamma irradiation at rates close to normal. Taken together these results indicate that: (1) DNA ligase I is involved only in nucleotide excision repair; (2) DNA ligase IV plays an important role only in repair of DNA double-strand breaks; and (3) DNA ligase III is implicated in base excision repair and in repair of DNA double-strand breaks, but probably not in nucleotide excision repair.

Animals↗
PubMed1976

Triacylglycerol synthesis in isolated fat cells. An effect of insulin on microsomal fatty acid coenzyme A ligase activity.

Fatty acid CoA ligase (AMP) (EC 6.2.1.3) specific activity was increased approximately 2-fold in microsomes prepared from isolated rat fat cells incubated with 400 microunits of insulin/ml (2.9 nM) for 45 to 60 min compared to paired controls using an assay based on the conversion of [3H]oleic acid to [3H]oleoyl-CoA. Similar insulin-dependent increases in microsomal fatty acid CoA ligase specific activities were observed using an assay based on the conversion of [3H]CoA to fatty acyl-[3H]CoA. Fatty acid CoA ligase activity was predominately (about 80%) associated with the microsomal fraction. The insulin-dependent increase in microsomal fatty acid CoA ligase specific activity was maximal in 2 to 5 min at 400 microunits/ml. At 10 min, 80 to 100 microunits of insulin/ml caused a maximal increase in fatty acid CoA ligase specific activity. Similar apparent Km values for ATP, CoA, and fatty acid were observed for fatty acid CoA ligase activity in microsomal preparations from control and insulin-exposed cells. These data suggest that fatty acid CoA ligase activity is regulated in adipose tissue by insulin. Such regulation may serve to promote the capture of fatty acid and thereby, triacylglycerol synthesis in adipose tissue.

Adenosine Triphosphate↗
PubMed1990

Purification and biochemical characterization of phenylacetyl-CoA ligase from Pseudomonas putida. A specific enzyme for the catabolism of phenylacetic acid.

A new enzyme, phenylacetyl-CoA ligase (AMP-forming) (PA-CoA ligase, EC 6.2.1-) involved in the catabolism of phenylacetic acid (PAA) in Pseudomonas putida is described and characterized. PA-CoA ligase was specifically induced by PAA when P. putida was grown in a chemically defined medium in which phenylacetic acid was the sole carbon source. Hydroxyl, methyl-phenylacetyl derivatives, and other PAA close structural molecules did not induce the synthesis of this enzyme and neither did acetic, butyric, succinic, nor fatty acids (greater than C5 atoms carbon length). PA-CoA ligase requires ATP, CoA, PAA, and MgCl2 for its activity. The maximal rate of catalysis was achieved in 50 mM HCl/Tris buffer, pH 8.2, at 30 degrees C and under these conditions, the Km calculated for ATP, CoA, and PAA were 9.7, 1.0, and 16.5 mM, respectively. The enzyme is inhibited by some divalent cations (Cu2+, Zn2+, and Hg2+) and by the sulfhydryl reagents N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and p-chloromercuribenzoate. PA-CoA ligase was purified to homogeneity (513-fold). It runs as a single polypeptide in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a molecular mass of 48 +/- 1 kDa. PA-CoA ligase does not use as substrate either 3-hydroxyphenylacetic, 4-hydroxyphenylacetic, or 3,4-dihydroxyphenylacetic acids and shows a substrate specificity different from other acyl-CoA-activating enzymes. The enzyme is detected in P. putida from the early logarithmic phase of growth and is repressed by glucose, suggesting that PA-CoA ligase is a specific enzyme involved in the utilization of PAA as energy source.

Amino Acids↗
PubMed2001

Muf1, a novel Elongin BC-interacting leucine-rich repeat protein that can assemble with Cul5 and Rbx1 to reconstitute a ubiquitin ligase.

The heterodimeric Elongin BC complex has been shown to interact in vitro and in mammalian cells with a conserved BC-box motif found in a growing number of proteins including RNA polymerase II elongation factor Elongin A, SOCS-box proteins, and the von Hippel-Lindau (VHL) tumor suppressor protein. Recently, the VHL-Elongin BC complex was found to interact with a module composed of Cullin family member Cul2 and RING-H2 finger protein Rbx1 to reconstitute a novel E3 ubiquitin ligase that activates ubiquitylation by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. In the context of the VHL ubiquitin ligase, Elongin BC functions as an adaptor that links the VHL protein to the Cul2/Rbx1 module, raising the possibility that the Elongin BC complex could function as an integral component of a larger family of E3 ubiquitin ligases by linking alternative BC-box proteins to Cullin/Rbx1 modules. In this report, we describe identification and purification from rat liver of a novel leucine-rich repeat-containing BC-box protein, MUF1, which we demonstrate is capable of assembling with a Cullin/Rbx1 module containing the Cullin family member Cul5 to reconstitute ubiquitin ligase activity. In addition, we show that the additional BC-box proteins Elongin A, SOCS1, and WSB1 are also capable of assembling with the Cul5/Rbx1 module to reconstitute potential ubiquitin ligases. Taken together, our findings identify MUF1 as a new member of the BC-box family of proteins, and they predict the existence of a larger family of Elongin BC-based E3 ubiquitin ligases.

Amino Acid Sequence↗
PubMed1990

Purification of DNA ligases from mouse testis and their behavior during meiosis.

Two types of DNA ligase, I and II, have been purified approximately 4,000-fold from mouse testes and 500-fold from nuclei of mouse spermatocytes. DNA ligase I and II consisted of single polypeptides with molecular weights of 95,000 and 65,000, respectively, according to the estimation by SDS-polyacrylamide gel electrophoresis and the AMP-binding assay. Ligase activities were higher in premeiotic spermatogonia and spermatocytes than those in liver and bone marrow cells. Moreover, DNA ligase II showed rapid increase during meiotic prophase and a decrease in round spermatids. Since this behavior of DNA ligase II is consistent with that of m-rec and DNA polymerase beta, both of which have been shown to be involved in DNA recombination in meiotic cells, DNA ligase II might be an enzyme which works at the final step of meiotic recombination reaction.

Adenosine Monophosphate↗
PubMed1986

Different substrate specificities of the two DNA ligases of mammalian cells.

Mammalian cells contain the DNA ligases I and II. These enzymes show different molecular weights and heat labilities, and antibodies against ligase I do not inhibit ligase II. Here, the nonidentical substrate specificities of the enzymes are described. Under standard reaction conditions DNA ligase I, but not ligase II, catalyzes blunt-end joining of DNA, while ligase II is the only activity that joins oligo(dT) molecules hydrogen-bonded to poly(rA). These differences facilitate the distinction between the two enzymes and should permit further analysis of their functions.

Animals↗
PubMed1993

Identity and some properties of the L-threonine aldolase activity manifested by pure 2-amino-3-ketobutyrate ligase of Escherichia coli.

2-Amino-3-ketobutyrate ligase catalyzes the reversible, pyridoxal 5'-phosphate-dependent condensation of glycine with acetyl CoA forming the unstable intermediate, 2-amino-3-ketobutyrate. Several independent lines of evidence indicate that the pure protein obtained in the purification of this ligase from Escherichia coli also has L-threonine aldolase activity. The evidence includes: (a), a constant ratio of specific activities (aldolase/ligase) at all stages of purifying 2-amino-3-ketobutyrate ligase to homogeneity; (b), the same rate of loss of aldolase and ligase activities during controlled heat inactivation of the pure protein at 60 degrees C in the absence, as well as in the presence of acetyl CoA, a protective substrate; (c), ratios of the two enzymatic activities that are not significantly different during slow inactivation by iodoacetamide, with and without L-threonine added; (d), coincident rates of loss and essentially identical rates of recovery of aldolase activity and ligase activity during resolution of the holoenzyme with hydroxylamine followed by reconstitution with pyridoxal 5'-phosphate. No aldolase activity is observed with D-threonine as substrate and L-allothreonine is about 25% as effective as L-threonine. Whereas ligase activity has a sharp pH optimum at 7.5, the aldolase activity of this pure protein is maximal at pH 9.0. Comparative apparent Km values for glycine (ligase) and L-threonine (aldolase) are 10 mM and 0.9 mM, respectively, whereas corresponding respective Vmax values were found to be 2.5 mumol of CoA released/min per mg vs. 0.014 mumol of acetaldehyde formed (NADH oxidized)/min per mg.

Acetyltransferases↗
PubMed2005

Quantitative assays of Mdm2 ubiquitin ligase activity and other ubiquitin-utilizing enzymes for inhibitor discovery.

Mdm2 is a negative regulator of p53 activity and functions as an E3 ubiquitin ligase of p53. Inhibition of mdm2 E3 ligase activity will block ubiquitination and subsequent proteasome-mediated degradation of p53, resulting in the stabilization of p53 protein that could lead to the restoration of its tumor-suppressor activity. This chapter describes quantitative biochemical assays for mdm2 E3 activity that can be applied to other ubiquitin-utilizing enzyme systems. Our unique assay format relies on the generation of labeled Ub-E2 conjugate that functions as a substrate for the E3 ligase enzyme. Reducing the E1-E2-E3 ubiquitin cascade to a single enzyme (E3) and bisubstrate (Ub-E2 and target protein) reaction makes it possible to carry out detailed biochemical characterization of the reaction mechanism, high-throughput screening to identify inhibitors of specific E3 ligases, and detailed characterization of the mode of inhibitor interactions with the target enzyme. In addition, preforming the Ub-E2 conjugate as an enzyme substrate for inhibitor screening minimizes interference from thiol-modifying compounds and from nucleotide analogs and other ATP-interfering compounds that might affect the E1 reaction. Using this type of format, we were able to identify small molecule inhibitors of mdm2 E3 ligase activity that are selective against E1 and other E3 ligases, including mdm2's own autoubiquitination activity. Detailed protocols on the labeling of Ub, the generation of Ub-E2, and the use of Ub-E2 in the E3 ligase reaction for inhibitor discovery and characterization are provided.

Drug Design↗
PubMed1996

Biochemical evidence for the formation of a covalent acyl-phosphate linkage between UDP-N-acetylmuramate and ATP in the Escherichia coli UDP-N-acetylmuramate:L-alanine ligase-catalyzed reaction.

In the peptidoglycan biosynthesis pathway in Escherichia coli, UDP-N-acetylmuramate:L-alanine ligase (MurC) catalyzes the formation of UDP-N-acetylmuramyl-L-alanine. A peptide bond is formed in this reaction and an ATP molecule is hydrolyzed concomitantly to produce ADP and orthophosphate. A biochemical approach was devised to elucidate the role of ATP in this reaction. A fusion construct pMAL::murC was prepared and the maltose binding protein--UDP-N-acetylmuramyl:L-alanine ligase fusion protein was overproduced in E. coli/pMal::murC upon isopropyl beta-thiogalactoside induction. The fusion protein was purified to > or = 90% homogeneity by a single-step affinity chromatography. Subsequently, the ligase was released from the maltose binding protein by proteolytic cleavage and was purified to > or = 95% homogeneity by an ion-exchange chromatographic step. The kinetic parameters of the regenerated ligase are comparable to those of the purified native enzyme. This ligase was used to investigate the role that ATP plays in the formation of UDP-N-acetylmuramyl-L-alanine. UDP-N-acetyl[18O]muramate (with 18O located at the carboxylate function only) was prepared by a combination of chemical and enzymatic processes and was used as the substrate of the ligase to probe the reaction mechanism. All reaction products were purified and subjected to liquid chromatographic-mass spectrometric analysis. A single [18O]oxygen was transferred from UDP-N-acetyl[18O]muramate to the orthophosphate produced in the reaction. No [18O]oxygen was detected in the adenosine nucleotides recovered from the reaction. These results strongly suggest that this ligase-catalyzed peptide formation proceeds through an activated acyl-phosphate linkage during the reaction process. ATP therefore assists in the process of the peptide bond formation by donating its gamma-phosphoryl group to activate the carboxyl group of UDP-N-acetylmuramic acid.

Adenosine Triphosphate↗
PubMed1997

D-Alanyl-D-lactate and D-alanyl-D-alanine synthesis by D-alanyl-D-alanine ligase from vancomycin-resistant Leuconostoc mesenteroides. Effects of a phenylalanine 261 to tyrosine mutation.

The Gram-positive bacterium Leuconostoc mesenteroides, ATCC 8293, is intrinsically resistant to the antibiotic vancomycin. This phenotype correlates with substitution of D-Ala-D-lactate (D-Ala-D-Lac) termini for D-Ala-D-Ala termini in peptidoglycan intermediates in which the depsipeptide has much lower affinity than the dipeptide for vancomycin binding. Overproduction of the L. mesenteroides D-Ala-D-Ala ligase (LmDdl) 2 in E. coli and its purification to approximately 90% homogeneity allow demonstration that the LmDdl2 does have both depsipeptide and dipeptide ligase activity. Recently, we reported that mutation of an active site tyrosine (Tyr), Tyr216, to phenylalanine (Phe) in the E. coli DdlB leads to gain of D-Ala-D-Lac depsipeptide ligase activity in that enzyme. The vancomycin-resistant LmDdl2 has a Phe at the equivalent site, Phe261. To test the prediction that a Tyr residue predicts dipeptide ligase while an Phe residue predicts both depsipeptide and dipeptide ligase activity, the F261Y mutant protein of LmDdl2 was constructed and purified to approximately 90% purity. F216Y LmDdl2 showed complete loss of the ability to couple D-Lac but retained D-Ala-D-Ala dipeptide ligase activity. The Tyr-->Phe substitution on the active site omega-loop in D-Ala-D-Ala ligases is thus a molecular indicator of both the ability to make D-Ala-D-Lac and intrinsic resistance to the vancomycin class of glycopeptide antibiotics.

Alanine↗
PubMed1994

Identification of the gene encoding lipoate-protein ligase A of Escherichia coli. Molecular cloning and characterization of the lplA gene and gene product.

R(+)-Lipoic acid is a cofactor required for function of the alpha-keto acid dehydrogenase and glycine cleavage enzyme complexes. The naturally occurring form of lipoate is attached by amide linkage to the epsilon-amino group of a specific lysine residue within conserved lipoate-accepting protein domains. Lipoate-protein ligase(s) catalyze the formation of this amide bond between lipoyl groups and specific apoproteins. We report the isolation of the lplA gene which encodes an Escherichia coli lipoate-protein ligase. Strains with lplA null mutations transport lipoic acid normally but have severe defects in the incorporation and utilization of exogenously supplied lipoic acid and lipoic acid analogs. These strains are also highly resistant to selenolipoate (a growth-inhibiting lipoate analog) and contain no detectable lipoate-protein ligase activity in cell extracts. The lplA gene has been cloned, sequenced, and physically mapped to min 99.6 (4657 kilobases) of the E. coli chromosome. Upon overexpression, the 38-kDa lplA gene product was purified to homogeneity and shown to have a mass, N-terminal sequence and amino acid composition consistent with the deduced 337 residue primary sequence. Enzyme assays show that purified LplA catalyzes the ATP-dependent attachment of [35S]lipoic acid to apoprotein, thus confirming that lplA encodes lipoate-protein ligase A. Analysis of lplA null mutants also indicates the existence of a second (lplA-independent) lipoyl-ligase enzyme in E. coli. This is the first identification of a lipoate ligase gene and the first analysis of a purified lipoate ligase enzyme.

Amino Acid Sequence↗