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At least 811 records · Page 45Linked to original sources
PubMed2000

The molecular basis of vancomycin resistance in clinically relevant Enterococci: crystal structure of D-alanyl-D-lactate ligase (VanA).

d-alanine-d-lactate ligase from Enterococcus faecium BM4147 is directly responsible for the biosynthesis of alternate cell-wall precursors in bacteria, which are resistant to the glycopeptide antibiotic vancomycin. The crystal structure has been determined with data extending to 2.5-A resolution. This structure shows that the active site has unexpected interactions and is distinct from previous models for d-alanyl-d-lactate ligase mechanistic studies. It appears that the preference of the enzyme for lactate as a ligand over d-alanine could be mediated by electrostatic effects and/or a hydrogen-bonding network, which principally involve His-244. The structure of d-alanyl-d-lactate ligase provides a revised interpretation of the molecular events that lead to vancomycin resistance.

Bacterial Proteins↗
PubMed1975

Induction of error-prone repair as a consequence of DNA ligase deficiency in Escherichia coli.

DNA ligase deficiency is shown to induce generalized mutator activity in E. coli. This mutator activity is unaffected by 3 mug/ml of chloramphenicol but is abolished both in lig-recA double mutants and by incubation with 20 mug/ml of chloramphenicol. Dna ligase deficiency is also shown to reactivate ultraviolet light-irradiated phage lambda and T7 and to increase both spontaneous and ultraviolet light-induced mutagenesis in phage lambda, all of which are abolished in lig-recA strains. Interaction occurs between the molecular events of mutagenesis induced by ultraviolet irradiation and those induced by DNA ligase deficiency. These observations suggest a common pathway, coordinately expressed with the inducible repair mode, that is responsible for mutagenesis.

Chloramphenicol↗
PubMed1986

Genetic definition of the translational operator of the threonine-tRNA ligase gene in Escherichia coli.

The Escherichia coli gene thrS that codes for threonine-tRNA ligase (tRNAThr ligase, formerly threonine-tRNA synthetase, EC 6.1.1.3) has previously been shown to be negatively autoregulated at the level of translation. Here we describe the use of several thrS-lac gene fusions to isolate cis-acting regulatory mutations that increase the translation but not the transcription of the thrS gene. These mutations lead to a total loss of control of repression and derepression of thrS. DNA sequence analysis locates the mutations between 10 and 40 base pairs upstream of the translation initiation codon of thrS and more than 100 base pairs downstream of the transcription initiation site. The mRNA region where these mutations are located shares primary and secondary structure homologies with specific parts of several isoacceptor tRNAThr species. These findings suggest that the ligase regulates its translation by binding to its mRNA at a place that shares some homology with its natural substrate.

Amino Acyl-tRNA Synthetases↗
PubMed1993

Cloning of the large subunit of activator 1 (replication factor C) reveals homology with bacterial DNA ligases.

We have cloned a gene encoding a DNA-binding protein by Southwestern screening of a murine cDNA library with a double-stranded oligonucleotide containing the sequence from the bidirectional promoter of the alpha 1 and alpha 2 collagen IV genes. The middle portion of this 1131-amino acid protein has a region homologous to bacterial DNA ligases, and the more carboxyl portion contains several domains homologous to p40, p38, p37, and p36.5 subunits of activator 1 (A1, also called replication factor C), a human replication protein complex. Western blotting revealed that antiserum generated against part of the recombinant protein reacted specifically with the 145-kDa component of the purified human A1 complex, indicating that it is the murine counterpart of the A1 p145. Characterization of the DNA-binding activity of the recombinant fusion protein by gel mobility-shift assay revealed that it had a preference for a run of pyrimidines on one strand. Deletion analysis using recombinant proteins revealed that the DNA ligase-like domain was required for DNA-binding activity. The finding that the region required for the binding of murine A1 p145 to DNA has similarity to a domain found in DNA ligases suggests that this region may be utilized by both proteins in recognizing DNA.

Amino Acid Sequence↗
PubMed1996

Trypanosoma brucei RNA editing. A full round of uridylate insertional editing in vitro mediated by endonuclease and RNA ligase.

RNA editing in kinetoplastids is the post-transcriptional insertion and deletion of uridylate residues in mitochondrial transcripts, directed by base pairing with guide RNAs. Models for editing propose transesterification or endonuclease plus RNA ligase reactions and may involve a guide RNA-mRNA chimeric intermediate. We have assessed the feasibility of the enzymatic pathway involving chimeras in vitro. Cytochrome b chimeras generated with mitochondrial extract were first found to have junctions primarily at the major endonuclease cleavage sites, supporting the role of endonuclease in chimera formation. Such cytochrome b chimeras are then specifically cleaved by extract endonuclease within the oligo(U) tract at the editing site, and the mRNA cleavage products are then joined by RNA ligase to generate partially edited mRNAs with uridylate residues transferred to an editing site. These in vitro generated partially edited mRNAs mimic partially edited mRNAs generated in vivo. Specific endonuclease cleavage in the editing region of the partially edited RNA demonstrates the potential for further in vitro editing. Finally, sensitivity to various ATP analogs suggests that all editing-like activities reported thus far utilize a mechanism involving RNA ligase.

Animals↗
PubMed1999

Standard free energy for the hydrolysis of adenylylated T4 DNA ligase and the apparent pKa of lysine 159.

Equilibrium constants for the adenylylation of T4 DNA ligase have been measured at 10 pH values. The values, when plotted against pH, fit a titration curve corresponding to a pKa of 8.4 +/- 0.1. The simplest interpretation is that the apparent pKa is that of the 6-amino group of the AMP-accepting residue Lys159. Based on the pH dependence of the equilibrium constants, the value at pH 7.0 is 0. 0213 at 25 degrees C, corresponding to DeltaG'o = +2.3 kcal mol-1. From this value and the standard free energy change of -10.9 kcal mol-1 for the hydrolysis of ATP to AMP and PPi, we calculate that DeltaG'o for the hydrolysis of the adenylyl-DNA ligase is -13.2 kcal mol-1. The presence of conserved basic amino acid residues in the catalytic domain, which are proximal to the active site in the homologous catalytic domain of T7 DNA ligase, suggests that the pKa of Lys159 is perturbed downward by the electrostatic effects of nearby positively charged amino acid side chains. The lower than normal pKa 8.4 compared with 10.5 for the 6-amino group of lysine and the high energy of the alpha,beta-phosphoanhydride linkage in ATP significantly facilitate adenylylation of the enzyme.

Adenosine Triphosphate↗
PubMed1999

The mitochondrial RNA ligase from Leishmania tarentolae can join RNA molecules bridged by a complementary RNA.

A biochemical characterization was performed with a partially purified RNA ligase from isolated mitochondria of Leishmania tarentolae. This ligase has a K(m) of 25 +/- 0.75 nM and a V(max) of 1.0 x 10(-4) +/- 2.4 x 10(-4) nmol/min when ligating a nicked double-stranded RNA substrate. Ligation was negatively affected by a gap between the donor and acceptor nucleotides. The catalytic efficiency of the circularization of a single-stranded substrate was 5-fold less than that of the ligation of a nicked substrate. These properties of the mitochondrial RNA ligase are consistent with an expected in vivo role in the process of uridine insertion/deletion RNA editing, in which the mRNA cleavage fragments are bridged by a cognate guide RNA.

Adenosine Triphosphate↗
PubMed2002

Deletion of a single amino acid residue from different 4-coumarate:CoA ligases from soybean results in the generation of new substrate specificities.

Plant 4-coumarate:coenzyme A ligases, acyl-CoA ligases, peptide synthetases, and firefly luciferases are grouped in one family of AMP-binding proteins. These enzymes do not only use a common reaction mechanism for the activation of carboxylate substrates but are also very likely marked by a similar functional architecture. In soybean, four 4-coumarate:CoA ligases have been described that display different substrate utilization profiles. One of these (Gm4CL1) represented an isoform that was able to convert highly ring-substituted cinnamic acids. Using computer-based predictions of the conformation of Gm4CL1, a peptide motif was identified and experimentally verified to exert a critical influence on the selectivity toward differently ring-substituted cinnamate substrates. Furthermore, one unique amino acid residue present in the other isoenzymes of soybean was shown to be responsible for the incapability to accommodate highly substituted substrates. The deletion of this residue conferred the ability to activate sinapate and, in one case, also 3,4-dimethoxy cinnamate and was accompanied by a significantly better affinity for ferulate. The engineering of the substrate specificity of the critical enzymes that activate the common precursors of a variety of phenylpropanoid-derived secondary metabolites may offer a convenient tool for the generation of transgenic plants with desirably modified metabolite profiles.

Amino Acid Sequence↗
PubMed2002

PIAS1 and PIASxalpha function as SUMO-E3 ligases toward androgen receptor and repress androgen receptor-dependent transcription.

The androgen receptor (AR) has been shown to be modified by SUMO-1, a ubiquitin-like protein. Recently we showed that PIAS family proteins function as SUMO-E3 ligases. Here we provide evidence that PIAS1 and PIASxalpha act as specific SUMO-E3 ligases for the AR. PIAS1 and PIASxalpha but not PIAS3 or PIASxbeta enhanced the sumoylation of AR in intact cells and in vitro. PIAS1 and PIASxalpha bound Ubc9, the E2 enzyme for SUMO-1, in a RING finger-like domain-dependent manner. Consistent with previous studies (Kahyo, T., Nishida, T., and Yasuda, H. (2001) Mol. Cell 8, 713-718), the RING finger-like domain of the SUMO-E3 was required for ligase activity. The binding of a ligand, e.g. testosterone, to the AR was required for the sumoylation of AR in intact cells. Although AR-dependent transcription was enhanced by PIAS proteins without sumoylation of the receptor, PIAS1 and PIASxalpha repressed AR-dependent transcription in a manner dependent on the ectopic expression of SUMO-1 and their RING finger-like domain. Furthermore, the sumoylation sites of the AR were necessary for the full repressive effect on AR-dependent transactivation, indicating that the sumoylation of AR was crucial for the repression of transactivation of the AR. Thus, PIAS1 and PIASxalpha modulate the AR-dependent transactivation, which, at least in part, can be attributed to their SUMO-E3 activity toward AR.

Humans↗
PubMed2003

Erythropoietin receptors associate with a ubiquitin ligase, p33RUL, and require its activity for erythropoietin-induced proliferation.

The proliferation and survival of hematopoietic cells is strictly regulated by cytokine growth factors that act through receptors of the Type I cytokine receptor family, including erythropoietin (Epo) and its receptor, EpoR. Mitogenic signaling by these receptors depends on activation of Jak tyrosine kinases. However, other required components of this pathway have not been fully identified. In a screen for proteins that interact with EpoR and Jak2, we identified a novel member of the U-box family of ubiquitin ligases. This receptor-associated ubiquitin ligase, RUL, co-precipitated with EpoR from mammalian cells and mediated ubiquitination of EpoR. Also, endogenously expressed RUL was rapidly and transiently phosphorylated on serine after cytokine treatment of factor-dependent hematopoietic cells. Expression of ubiquitin ligase-deficient mutants of RUL inhibited Epo-induced expression of c-myc and bcl-2, two immediate-early genes normally associated with Epo-induced cell growth. Consistent with that finding, expression of mutant RUL also inhibited Epo-dependent proliferation and survival of factor-dependent cells. Together, these observations suggest that RUL is a required component of mitogenic signaling by EpoR. We also show that RUL is phosphorylated in response to growth factors that act through non-cytokine receptors, suggesting that RUL may function as a common regulator of mitogenesis.

Amino Acid Sequence↗
PubMed2003

Human HRD1 is an E3 ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum.

The ubiquitin system plays an important role in endoplasmic reticulum (ER)-associated degradation of proteins that are misfolded, that fail to associate with their oligomerization partners, or whose levels are metabolically regulated. E3 ubiquitin ligases are key enzymes in the ubiquitination process as they recognize the substrate and facilitate coupling of multiple ubiquitin units to the protein that is to be degraded. The Saccharomyces cerevisiae ER-resident E3 ligase Hrd1p/Der3p functions in the metabolically regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and additionally facilitates the degradation of a number of misfolded proteins from the ER. In this study we characterized the structure and function of the putative human orthologue of yeast Hrd1p/Der3p, designated human HRD1. We show that human HRD1 is a non-glycosylated, stable ER protein with a cytosolic RING-H2 finger domain. In the presence of the ubiquitin-conjugating enzyme UBC7, the RING-H2 finger has in vitro ubiquitination activity for Lys(48)-specific polyubiquitin linkage, suggesting that human HRD1 is an E3 ubiquitin ligase involved in protein degradation. Human HRD1 appears to be involved in the basal degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase but not in the degradation that is regulated by sterols. Additionally we show that human HRD1 is involved in the elimination of two model ER-associated degradation substrates, TCR-alpha and CD3-delta.

Amino Acid Sequence↗
PubMed2004

RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2.

Here we report that bacteriophage T4 RNA ligase 2 (Rnl2) is an efficient catalyst of RNA ligation at a 3'-OH/5'-PO(4) nick in a double-stranded RNA or an RNA.DNA hybrid. The critical role of the template strand in approximating the reactive 3'-OH and 5'-PO(4) termini is underscored by the drastic reductions in the RNA-sealing activity of Rnl2 when the duplex substrates contain gaps or flaps instead of nicks. RNA nick joining requires ATP and a divalent cation cofactor (either Mg or Mn). Neither dATP, GTP, CTP, nor UTP can substitute for ATP. We identify by alanine scanning seven functionally important amino acids (Tyr-5, Arg-33, Lys-54, Gln-106, Asp-135, Arg-155, and Ser-170) within the N-terminal nucleotidyl-transferase domain of Rnl2 and impute specific roles for these residues based on the crystal structure of the AMP-bound enzyme. Mutational analysis of 14 conserved residues in the C-terminal domain of Rnl2 identifies 3 amino acids (Arg-266, Asp-292, and Glu-296) as essential for ligase activity. Our findings consolidate the evolutionary connections between bacteriophage Rnl2 and the RNA-editing ligases of kinetoplastid protozoa.

Amino Acid Sequence↗
PubMed2004

Ubiquitylation of neuronal nitric-oxide synthase by CHIP, a chaperone-dependent E3 ligase.

It is established that neuronal nitric-oxide synthase (nNOS) is ubiquitylated and proteasomally degraded. The proteasomal degradation of nNOS is enhanced by suicide inactivation of nNOS or by the inhibition of hsp90, which is a chaperone found in a native complex with nNOS. In the current study, we have examined whether CHIP, a chaperone-dependent E3 ubiquitin-protein isopeptide ligase that is known to ubiquitylate other hsp90-chaperoned proteins, could act as an ubiquitin ligase for nNOS. We found with the use of HEK293T or COS-7 cells and transient transfection methods that CHIP overexpression causes a decrease in immunodetectable levels of nNOS. The extent of the loss of nNOS is dependent on the amount of CHIP cDNA used for transfection. Lactacystin (10 microM), a selective proteasome inhibitor, attenuates the loss of nNOS in part by causing the nNOS to be found in a detergent-insoluble form. Immunoprecipitation of the nNOS and subsequent Western blotting with an anti-ubiquitin IgG shows an increase in nNOS-ubiquitin conjugates because of CHIP. Moreover, incubation of nNOS with a purified system containing an E1 ubiquitin-activating enzyme, an E2 ubiquitin carrier protein conjugating enzyme (UbcH5a), CHIP, glutathione S-transferase-tagged ubiquitin, and an ATP-generating system leads to the ubiquitylation of nNOS. The addition of purified hsp70 and hsp40 to this in vitro system greatly enhances the amount of nNOS-ubiquitin conjugates, suggesting that CHIP is an E3 ligase for nNOS whose action is facilitated by (and possibly requires) its interaction with nNOS-bound hsp70.

Acetylcysteine↗
PubMed2004

An RNA ligase from Deinococcus radiodurans.

Although DNA repair pathways have been the focus of much attention, there is an emerging appreciation that distinct pathways exist to maintain or manipulate RNA structure in response to breakage events. Here we identify an RNA ligase (DraRnl) from the radiation-resistant bacterium Deinococcus radiodurans. DraRnl seals 3'-OH/5'-PO4 RNA nicks in either a duplex RNA or an RNA: DNA hybrid, but it cannot seal 3'-OH/5'-PO4 DNA nicks. The specificity of DraRnl arises from a requirement for RNA on the 3'-OH side of the nick. DraRnl is a 342-amino acid monomeric protein with a distinctive structure composed of a C-terminal adenylyltransferase domain linked to an N-terminal module that resembles the OB-fold of phenylalanyl-tRNA synthetases. RNA sealing activity was abolished by mutation of the predicted lysine adenylylation site (Lys-165) in the C-terminal domain and was reduced by an order of magnitude by deletion of the N-terminal OB module. Our findings highlight the existence of an RNA repair capacity in bacteria and support the hypothesis that contemporary DNA ligases, RNA ligases, and RNA capping enzymes evolved by the fusion of ancillary effector domains to an ancestral catalytic module involved in RNA repair.

Alternative Splicing↗
PubMed2005

Structure-guided mutational analysis of the nucleotidyltransferase domain of Escherichia coli NAD+-dependent DNA ligase (LigA).

NAD+-dependent DNA ligase (LigA) is essential for bacterial growth and a potential target for antimicrobial drug discovery. Here we queried the role of 14 conserved amino acids of Escherichia coli LigA by alanine scanning and thereby identified five new residues within the nucleotidyltransferase domain as being essential for LigA function in vitro and in vivo. Structure activity relationships were determined by conservative mutagenesis for the Glu-173, Arg-200, Arg-208, and Arg-277 side chains, as well as four other essential side chains that had been identified previously (Lys-115, Asp-117, Asp-285, and Lys-314). In addition, we identified Lys-290 as important for LigA activity. Reference to the structure of Enterococcus faecalis LigA allowed us to discriminate three classes of essential/important side chains that: (i) contact NAD+ directly (Lys-115, Glu-173, Lys-290, and Lys-314); (ii) comprise the interface between the NMN-binding domain (domain Ia) and the nucleotidyltransferase domain or comprise part of a nick-binding site on the surface of the nucleotidyltransferase domain (Arg-200 and Arg-208); or (iii) stabilize the active site fold of the nucleotidyltransferase domain (Arg-277). Analysis of mutational effects on the isolated ligase adenylylation and phosphodiester formation reactions revealed different functions for essential side chains at different steps of the DNA ligase pathway, consistent with the proposal that the active site is serially remodeled as the reaction proceeds.

Alanine↗
PubMed2005

The chaperone-associated ubiquitin ligase CHIP is able to target p53 for proteasomal degradation.

The cellular level of the tumor suppressor p53 is tightly regulated through induced degradation via the ubiquitin/proteasome system. The ubiquitin ligase Mdm2 plays a pivotal role in stimulating p53 turnover. However, recently additional ubiquitin ligases have been identified that participate in the degradation of the tumor suppressor. Apparently, multiple degradation pathways are employed to ensure proper destruction of p53. Here we show that the chaperone-associated ubiquitin ligase CHIP is able to induce the proteasomal degradation of p53. CHIP-induced degradation was observed for mutant p53, which was previously shown to associate with the chaperones Hsc70 and Hsp90, and for the wild-type form of the tumor suppressor. Our data reveal that mutant and wild-type p53 transiently associate with molecular chaperones and can be diverted onto a degradation pathway through this association.

Cell Line↗
PubMed2005

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.

Acyltransferases↗
PubMed2006

The viral E3 ubiquitin ligase mK3 uses the Derlin/p97 endoplasmic reticulum-associated degradation pathway to mediate down-regulation of major histocompatibility complex class I proteins.

Ubiquitin E3 ligases are important cellular components for endoplasmic reticulum (ER)-associated degradation due to their role in substrate-specific ubiquitination, which is required for retrotranslocation (dislocation) of most unwanted proteins from the ER to the cytosol for proteasome degradation. However, our understanding of the molecular mechanisms of how E3 ligases confer substrate-specific recognition, and their role in substrate retrotranslocation is limited especially in mammalian cells. mK3 is a type III ER membrane protein encoded by murine gamma herpesvirus 68. As conferred by its N-terminal RING-CH domain, mK3 has E3 ubiquitin ligase activity. In its role as an immune evasion protein, mK3 specifically targets nascent major histocompatibility complex class I heavy chains (HC) for rapid degradation. The mechanism by which mK3 extracts HC from the ER membrane into the cytosol for proteasome-mediated degradation is unknown. Evidence is presented here that HC down-regulation by mK3 is dependent on the p97 AAA-ATPase. By contrast, the kK5 protein of Kaposi's sarcoma-associated herpesvirus is p97-independent despite the fact that it is highly homologous to mK3. mK3 protein was also found in physical association with Derlin1, an ER protein recently implicated in the retrotranslocation of HC by immune evasion protein US11, but not US2, of human cytomegalovirus. The mechanistic implications of these findings are discussed.

Adenosine Triphosphatases↗