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At least 1,369 records · Page 76Linked to original sources
PubMed1999

Mutational analysis of active-site residues of the enterococcal D-ala-D-Ala dipeptidase VanX and comparison with Escherichia coli D-ala-D-Ala ligase and D-ala-D-Ala carboxypeptidase VanY.

BACKGROUND: Vancomycin-resistant enterococci are pathogenic bacteria that attenuate antibiotic sensitivity by producing peptidoglycan precursors that terminate in D-Ala-D-lactate rather than D-Ala-D-Ala. A key enzyme in effecting antibiotic resistance is the metallodipeptidase VanX, which reduces the cellular pool of the D-Ala-D-Ala dipeptide. RESULTS: We constructed eleven mutants, using the recently determined VanX structure as a basis, to investigate residue function. Mutating Asp142 or Ser114 showed a large effect principally on KM, consistent with roles in recognition of the D-Ala-D-Ala termini. The drastic reduction or absence of activity in the Arg71 mutants correlates with a role in the stabilization of an anionic tetrahedral transition state. Three residues of the Escherichia coli D-Ala-D-Ala ligase (Ddl), Glu15, Ser 281 and Arg255, are similarly conserved and have equivalent functions with respect to VanX, consistent with a convergent evolution of active sites to bind D-Ala-D-Ala and lower energy barriers for formation of the tetrahedral intermediate and transition states. In the N-acyl-D-Ala-D-Ala carboxypeptidase VanY, all active-site residues are conserved (except for the two responsible for recognition of the dipeptide amino terminus). CONCLUSIONS: The mutagenesis results support structure-based functional predictions and explain why the VanX dipeptidase and Ddl ligase show narrow specificity for the D,D-dipeptide substrate. The results reveal that VanX and Ddl, two enzymes that use the same substrate but proceed in opposite directions driven by distinct cofactors (zinc versus ATP), evolved similar architectural solutions to substrate recognition and catalysis acceleration. VanY sequence analysis predicts an active site and mechanism of reaction similar to VanX.

Anti-Bacterial Agents↗
PubMed2004

The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes.

The CARD domain protein BCL10 and paracaspase MALT1 are essential for the activation of IkappaB kinase (IKK) and NF-kappaB in response to T cell receptor (TCR) stimulation. Here we present evidence that TRAF6 ubiquitin ligase and TAK1 protein kinase mediate IKK activation by BCL10 and MALT1. RNAi-mediated silencing of MALT1, TAK1, TRAF6, and TRAF2 suppressed TCR-dependent IKK activation and interleukin-2 production in T cells. Furthermore, we have reconstituted the pathway from BCL10 to IKK activation in vitro with purified proteins of MALT1, TRAF6, TAK1, and ubiquitination enzymes including Ubc13/Uev1A. We find that a small fraction of BCL10 and MALT1 proteins form high molecular weight oligomers. Strikingly, only these oligomeric forms of BCL10 and MALT1 can activate IKK in vitro. The MALT1 oligomers bind to TRAF6, induce TRAF6 oligomerization, and activate the ligase activity of TRAF6 to polyubiquitinate NEMO. These results reveal an oligomerization --> ubiquitination --> phosphorylation cascade that culminates in NF-kappaB activation in T lymphocytes.

Adaptor Proteins, Signal Transducing↗
PubMed2003

CHIP: a quality-control E3 ligase collaborating with molecular chaperones.

It is notable that both the chaperone and ubiquitin-proteasome systems are required for removal of aberrant cellular proteins to ensure protein homeostasis in cells. However, the entity that links the two systems had remained elusive. Carboxyl-terminus of Hsc70 interacting protein (CHIP), originally identified as a co-chaperone of Hsc70, has both a tetratricopeptide repeat (TPR) motif and a U-box domain. The TPR motif associates with Hsc70 and Hsp90, while the U-box domain executes a ubiquitin ligase activity. Thus, CHIP is an ideal molecule acting as a protein quality-control ubiquitin ligase that selectively leads abnormal proteins recognized by molecular chaperones to degradation by the proteasome. Accumulating evidence from in vitro studies indicates that this is apparently the case. Here, we present and discuss several unresolved but critical issues related to the molecular mechanism and in vivo roles of CHIP.

Animals↗
PubMed1991

Existence of two D-alanine:D-alanine ligases in Escherichia coli: cloning and sequencing of the ddlA gene and purification and characterization of the DdlA and DdlB enzymes.

Two distinct genes encoding D-alanine:D-alanine (D-Ala-D-Ala) ligase (ADP forming) activity in Escherichia coli have been cloned by complementation of E. coli strain ST640(lambda 112) deficient in D-Ala-D-Ala ligase activity with a lambda library of E. coli DNA. One of the two genes, designated as ddlB, is identical with the ddl gene already sequenced [Robinson, A.C., Kenan, D.L., Sweeney, J., & Donachie, W.D. (1986) J. Bacteriol. 167, 809-817]. We describe the subcloning and DNA sequencing of the other gene, designated as ddlA on the basis of similarities with the Salmonella typhimurium ddlA gene [Daub, E., Zawadzke, L.E., Botstein, D., & Walsh, C.T. (1988) Biochemistry 27, 3701-3708]. The predicted amino acid sequence of the E. coli DdlA enzyme shows 90% homology with the S. typhimurium DdlA sequence. The ddlB gene was subcloned by use of the polymerase chain reaction into an expression vector containing an optimized ribosome binding site, which expressed the DdlB enzyme to greater than 50% soluble cell protein. Both DdlA and DdlB enzymes were purified to greater than 90% homogeneity and characterized kinetically.

Amino Acid Sequence↗
PubMed1988

Isolation, cloning, and sequencing of the Salmonella typhimurium ddlA gene with purification and characterization of its product, D-alanine:D-alanine ligase (ADP forming).

A gene coding for D-alanine:D-alanine (D-Ala-D-Ala) ligase (ADP forming) (EC 6.3.2.4) activity has been isolated from a lambda library of Salmonella typhimurium DNA. Insertion mutations in the gene indicate that the gene is not essential for growth of the bacterium. The encoded enzyme was purified from an overproducing strain of S. typhimurium. D-Ala-D-Ala ligase is a protein of 39,271 molecular weight and has a kcat of 644 min-1 at pH 7.2. A 2.4-kilobase SalI-SphI fragment containing the gene was sequenced, and the ddlA gene consists of 1092 nucleotides. The gene sequence was compared to the sequence of the ddl gene of Escherichia coli [Robinson, A. C., Kenan, D. J., Sweeney, J., & Donachie, W. D. (1986) J. Bacteriol. 167, 809-817]. Because of differences between the S. typhimurium gene and the E. coli ddl gene, the S. typhimurium gene has been named ddlA.

Amino Acid Sequence↗
PubMed1989

(1-Aminoethyl)boronic acid: a novel inhibitor for Bacillus stearothermophilus alanine racemase and Salmonella typhimurium D-alanine:D-alanine ligase (ADP-forming).

(1-Aminoethyl)boronic acid (Ala-B), an analogue of alanine in which a boronic acid group replaces the carboxyl group, has been synthesized and found to inhibit the first two enzymes, alanine racemase (from Bacillus stearothermophilus, EC 5.1.1.1) and D-alanine:D-alanine ligase (ADP-forming) (from Salmonella typhimurium, EC 6.3.2.4), of the D-alanine branch of bacterial peptidoglycan biosynthesis. In both cases, time-dependent, slow binding inhibition is observed due to the generation of long-lived, slowly dissociating complexes. Ala-B inhibits alanine racemase with a Ki of 20 mM and a kappa inact of 0.15-0.35 min-1. Time-dependent loss of activity is paralleled by conversion of the 420-nm chromophore of initial bound PLP aldimine to a 324-nm absorbing species. On dilution of Ala-B, racemase activity is regained with a t1/2 of ca. 1 h. The D-Ala-D-Ala ligase also shows progressive inhibition by Ala-B provided ATP (but not AMP-PNP or AMP-PCP) is present. The presence of D-alanine along with ATP also leads to Ala-B-induced inactivation. Kinetic analysis suggests Ala-B can compete with D-alanine at either of the two D-alanine binding sites, and on inactivation with Ala-B, labeled D-alanine, and labeled ATP, the inactive enzyme has stoichiometric amounts of D-alanine, ADP, Pi, and Ala-B bound. The half-life of inactive enzyme complexes varied from approximately 2 h (without D-alanine) to 4.5 days (with D-alanine). No D-Ala-D-Ala-B dipeptide was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1990

Rotational resonance determination of the structure of an enzyme-inhibitor complex: phosphorylation of an (aminoalkyl)phosphinate inhibitor of D-alanyl-D-alanine ligase by ATP.

We have used a newly developed solid-state NMR method, rotational resonance, to establish the structure of an inhibited complex formed upon reaction of D-alanyl-D-alanine ligase, ATP, and the aminoalkyl dipeptide analogue [1(S)-aminoethyl][2-carboxy-2(R)-methyl-1- ethyl]phosphinic acid (Ib). Analogue Ib was determined to be an ATP-dependent, slow-binding inhibitor of the D-Ala-D-Ala ligase from Salmonella typhimurium, with an enzyme-inhibitor half-life of 17 days at 37 degrees C. The inhibited complex shows a 31P NMR spectrum which is very different from that which would arise from a mixture of the free inhibitor and ATP. Four well-resolved lines were observed: two (at -8 and -14 ppm) are assignable as the phosphates of ADP, the third is assignable to an inhibitor resonance (at 53 ppm) that shifts by approximately 19 ppm on binding, and the fourth is assignable to a resonance (at -3 ppm) due to a polyphosphate or phosphate ester moiety. At rotational resonance the spectrum shows evidence for strong dipolar couplings between the phosphinate phosphorus and a phosphate ester species. The dipolar coupling between the phosphorus signals at 53 and -3 ppm was measured at rotational resonance by use of numerical simulations of both the line shape of the signal and the profile of magnetization transfer between the two sites. The measured coupling, 1.0 +/- 0.2 kHz, indicates that the two species are bridged in a P-O-P linkage, with a P-P through-space distance of 2.7 +/- 0.2 A. This proves that the mechanism of inactivation involves phosphorylation of the enzyme-bound inhibitor by ATP to form a phosphoryl-phosphinate adduct.

Adenosine Triphosphate↗
PubMed1997

Evaluation of the kinetic mechanism of Escherichia coli uridine diphosphate-N-acetylmuramate:L-alanine ligase.

Initial velocity methods were used to probe the kinetic mechanism of Escherichia coli uridine diphosphate-N-acetylmuramate:L-alanine ligase (UNAM:L-Ala ligase). When the activity (in the forward direction) versus substrate concentration data were plotted in double-reciprocal form, all line patterns were intersecting. The best fit of these data was to the equation for an ordered mechanism with the following parameters: k(cat), 1000 +/- 100 min(-1); Kma, 210 +/- 40 microM; Kmb, 84 +/- 20 microM; Kmc, 70 +/- 15 microM; Kia, 180 +/- 50 microM; Kib, 68 +/- 24 microM. Initial velocity line patterns were also determined when the concentration of one substrate was varied at different fixed concentrations of a second substrate while the third substrate was held at a concentration more than 100 times its Km value. Reciprocal plots of data collected with either ATP or L-alanine present at more than 100 times their Km values resulted in intersecting line patterns. Data collected with UNAM present at 100 times its Km value gave a set of parallel lines. These data are consistent with UNAM binding as the second substrate in an ordered mechanism. ADP, uridine diphosphate-N-acetylmuramoyl-L-alanine (UNAMA), and phosphate were tested as product inhibitors versus substrates. None of the products were competitive inhibitors versus L-alanine or UNAM, while the only observed competitive inhibition was ADP versus ATP. These results are consistent with an ordered kinetic mechanism wherein ATP binds first, UNAM binds second, and ADP is the last product released. Rapid quench experiments were performed in the presence of all three substrates or in the presence of ATP and UNAM. The production of acid-labile phosphate as a function of time is characterized by a burst phase followed by a slower linear phase with the rate close to k(cat) in the presence of all three substrates. Only a burst phase was observed for the time course of the reaction in the presence of ATP and UNAM. In both cases, the burst rate was identical. These observations are consistent with L-alanine being the third substrate to bind in a sequential mechanism involving a putative acyl-phosphate intermediate.

Adenosine Triphosphate↗
PubMed1986

(beta-Chloro-alpha-aminoethyl)phosphonic acids as inhibitors of alanine racemase and D-alanine:D-alanine ligase.

The (beta-chloro-, (beta, beta-dichloro-, and (beta, beta, beta-trichloro-alpha-aminoethyl)phosphonic acids have been synthesized and their inhibitory properties on the alanine racemases [EC 5.1.1.1] and the D-Ala:D-Ala ligases [EC 6.3.2.4] from Pseudomonas aeruginosa and Streptococcus faecalis have been evaluated. The monochloro and the dichloro derivatives of Ala-P exhibit a strong inhibition on the racemases of the two species tested but do not behave as suicide substrates. Only the D-Ala:D-Ala ligase of S. faecalis is inhibited by these compounds. The poor antibacterial activity observed with beta-chloro- and beta, beta-dichloro-Ala-P might be enhanced by the peptide-transport strategy.

Alanine Racemase↗
PubMed1999

Calculation of the pKa values for the ligands and side chains of Escherichia coli D-alanine:D-alanine ligase.

Poisson-Boltzmann electrostatics methods have been used to calculate the pKa shifts for the ligands and titratable side chains of D-alanine:D-alanine ligase of the ddlb gene of Escherichia coli (DdlB). The focus of this study is to determine the ionization state of the second D-alanine (D-Ala2) in the active site of DdlB. The pKa of the amine is shifted over 5 pKa units more alkaline in the protein, clearly implying that D-Ala2 is bound to DdlB in its zwitterionic state and not in the free-base form as had been previously suggested. Comparisons are made to the depsipeptide ligase from the vancomycin-resistance cascade, VanA. It is suggested that VanA has different enzymatic properties due to a change in binding specificity rather than altered catalytic behavior and that the specificity of binding D-lactate over D-Ala2 may arise from the difference in ionization characteristics of the ligands.

Binding Sites↗
PubMed2003

The COP9 signalosome: an assembly and maintenance platform for cullin ubiquitin ligases?

The COP9 signalosome (CSN) is a highly conserved protein complex implicated in diverse biological functions that involve ubiquitin-mediated proteolysis. Paradoxically, conserved enzymatic activities associated with CSN inhibit cullin ubiquitin ligase activity in vitro, whereas mutational analysis suggests that CSN promotes cullin-dependent proteolysis in vivo. This apparent paradox can be resolved in a model that proposes CSN-mediated cullin inhibition is a prerequisite for the proper assembly and maintenance of active cullin ubiquitin ligase complexes.

Animals↗
PubMed2005

Src-like adaptor protein regulates TCR expression on thymocytes by linking the ubiquitin ligase c-Cbl to the TCR complex.

The adaptor molecule SLAP and E3 ubiquitin ligase c-Cbl each regulate expression of T cell receptor (TCR)-CD3 on thymocytes. Here we provide genetic and biochemical evidence that both molecules function in the same pathway. TCR-CD3 expression was similar in the absence of SLAP and/or c-Cbl. SLAP and c-Cbl were found to interact, and their expression together downregulated CD3epsilon. This required multiple domains in SLAP and the ring finger of c-Cbl. Furthermore, expression of SLAP and c-Cbl together induced TCRzeta ubiquitination and degradation, preventing the accumulation of fully assembled recycling TCR complexes. These studies indicate that SLAP links the E3 ligase activity of c-Cbl to the TCR, allowing for stage-specific regulation of TCR expression.

Animals↗
PubMed2006

Mechanism and substrate stereochemistry of 2-amino-3-oxobutyrate CoA ligase: implications for 5-aminolevulinate synthase and related enzymes.

The condensation process catalysed by 2-amino-3-oxobutyrate CoA ligase (KBL; also known as 2-amino-3-ketobutyrate ligase) involves the loss of the pro-R hydrogen atom of glycine and occurs with the inversion of stereochemistry; a similar scenario is envisaged for the condensation step of other alpha-oxoamine synthases.

5-Aminolevulinate Synthetase↗
PubMed2003

Negative regulation of Lyn protein-tyrosine kinase by c-Cbl ubiquitin-protein ligase in Fc epsilon RI-mediated mast cell activation.

BACKGROUND: Recent studies have demonstrated that c-Cbl functions as a ubiquitin-protein ligase toward immune receptors and non-receptor protein-tyrosine kinase Syk by facilitating their ubiquitination and subsequent targeting to proteasomes. However, it was not clear whether Src family kinase Lyn is regulated by the Cbl family of ubiquitin-protein ligases. RESULTS: Aggregation of the high affinity IgE receptor (Fc epsilon RI) induces the rapid ubiquitination of Lyn in rat basophilic leukaemia RBL-2H3 cells. Treatment of cells with a proteasome inhibitor enhances the ubiquitination of Lyn. Stimulation of Fc epsilon RI results in the association of Lyn with c-Cbl and Cbl-b, both of which then become tyrosine phosphorylated. Co-transfection study shows that both c-Cbl and Cbl-b could induce the ubiquitination of activated Lyn in COS cells. Furthermore, over-expression of membrane-anchored form of c-Cbl inhibits the Fc epsilon RI-mediated degranulation and cytokine gene production in RBL-2H3 cells by the down-regulation of the kinase activity of Lyn through the enhanced ubiquitination. CONCLUSIONS: These results demonstrate that Lyn is down-regulated by c-Cbl-mediated ubiquitination and subsequent degradation in proteasome after Fc epsilon RI stimulation in mast cells. Targeting of c-Cbl in the lipid raft results in the inhibition of Fc epsilon RI-mediated mast cell activation.

Animals↗
PubMed2006

Allosteric inhibition of Staphylococcus aureus D-alanine:D-alanine ligase revealed by crystallographic studies.

D-alanine:D-alanine ligase (DDl) is an essential enzyme in bacterial cell wall biosynthesis and an important target for developing new antibiotics. It catalyzes the formation of D-alanine:D-alanine dipeptide, sequentially by using one D-alanine and one ATP as substrates for the first-half reaction, and a second D-alanine substrate to complete the reaction. Some gain of function DDl mutants can use an alternate second substrate, causing resistance to vancomycin, one of the last lines of defense against life-threatening Gram-positive infections. Here, we report the crystal structure of Staphylococcus aureus DDl (StaDDl) and its cocrystal structures with 3-chloro-2,2-dimethyl-N-[4(trifluoromethyl)phenyl]propanamide (inhibitor 1) (Ki=4 microM against StaDDl) and with ADP, one of the reaction products, at resolutions of 2.0, 2.2, and 2.6 A, respectively. The overall structure of StaDDl can be divided into three distinct domains. The inhibitor binds to a hydrophobic pocket at the interface of the first and the third domain. This inhibitor-binding pocket is adjacent to the first D-alanine substrate site but does not overlap with any substrate sites. An allosteric inhibition mechanism of StaDDl by this compound was proposed. The mechanism provides the basis for developing new antibiotics targeting D-alanine:D-alanine ligase. Because this compound only interacts with residues from the first D-alanine site, inhibitors with this binding mode potentially could overcome vancomycin resistance.

Allosteric Regulation↗
PubMed1994

Covalent catalysis in nucleotidyl transfer reactions: essential motifs in Saccharomyces cerevisiae RNA capping enzyme are conserved in Schizosaccharomyces pombe and viral capping enzymes and among polynucleotide ligases.

Formation of the 5' cap structure of eukaryotic mRNAs occurs via transfer of GMP from GTP to the 5' terminus of the primary transcript. RNA guanylyltransferase, the enzyme that catalyzes this reaction, has been isolated from many viral and cellular sources. Though differing in molecular weight and subunit structure, the various guanylyltransferases employ a common catalytic mechanism involving a covalent enzyme-(Lys-GMP) intermediate. Saccharomyces cerevisiae CEG1 is the sole example of a cellular capping enzyme gene. In this report, we describe the identification and characterization of the PCE1 gene encoding the capping enzyme from Schizosaccharomyces pombe. PCE1 was isolated from a cDNA library by functional complementation in Sa. cerevisiae. Induced expression of PCE1 in bacteria and in yeast confirmed that the 47-kDa Sc. pombe protein was enzymatically active. The amino acid sequence of PCE1 is 38% identical (152 of 402 residues) to the 52-kDa capping enzyme from Sa. cerevisiae. Comparison of the two cellular capping enzymes with guanylyltransferases encoded by DNA viruses revealed local sequence similarity at the enzyme's active site and at four additional collinear motifs. Mutational analysis of yeast CEG1 demonstrated that four of the five conserved motifs are essential for capping enzyme function in vivo. Remarkably, the same motifs are conserved in the polynucleotide ligase family of enzymes that employ an enzyme-(Lys-AMP) intermediate. These findings illuminate a shared structural basis for covalent catalysis in nucleotidyl transfer and suggest a common evolutionary origin for capping enzymes and ligases.

Amino Acid Sequence↗
PubMed2006

Biotin sensing in Saccharomyces cerevisiae is mediated by a conserved DNA element and requires the activity of biotin-protein ligase.

Biotin is a water-soluble vitamin that functions as a prosthetic group in carboxylation reactions. In addition to its role as a cofactor, biotin has multiple roles in gene regulation. We analyzed biotin effects on gene expression in the yeast Saccharomyces cerevisiae and demonstrated by microarray, Northern, and Western analyses that all yeast genes encoding proteins involved in biotin metabolism are up-regulated following biotin depletion. Many of these genes contain a palindromic promoter element that is necessary and sufficient for mediating the biotin response and functions as an upstream-activating sequence. Mutants lacking the plasma membrane biotin transporter Vht1p display constitutively high expression levels of biotin-responsive genes. However, they react normally to biotin precursors that do not require Vht1p for uptake. The biotin-like effect of precursors with regard to gene expression requires their intracellular conversion to biotin. This demonstrates that Vht1p does not act as a sensor for biotin and that intracellular biotin is crucial for gene expression. Mutants with defects in biotin-protein ligase, similar to vht1delta mutants, also display aberrantly high expression of biotin-responsive genes. Like vht1delta cells, they have reduced levels of protein biotinylation, but unlike vht1delta mutants, they possess normal levels of free intracellular biotin. This indicates that free intracellular biotin is irrelevant for gene regulation and identifies biotin-protein ligase as an important element of the biotin-sensing pathway in yeast.

Base Sequence↗
PubMed1996

Study of the overproduced uridine-diphosphate-N-acetylmuramate:L-alanine ligase from Escherichia coli.

The UDP-N-acetylmuramate:L-alanine ligase of Escherichia coli is responsible for the addition of the first amino acid of the peptide moiety in the assembly of the monomer unit of peptidoglycan. It catalyzes the formation of the amide bond between UDP-N-acetylmuramic acid (UDP-MurNAc) and L-alanine. The UDP-MurNAc-L-alanine ligase was overproduced 2000-fold in a strain harboring a recombinant plasmid (pAM1005) with the murC gene under the control of the inducible promoter trc. The murC gene product appears as a 50-kDa protein accounting for ca. 50% of total cell proteins. A two-step purification led to 1 g of a homogeneous protein from an 8-liter culture. The N-terminal sequence of the purified protein correlated with the nucleotide sequence of the gene. The stability of the enzymatic activity is strictly dependent on the presence of 2-mercaptoethanol. The K(m) values for substrates UDP-N-acetylmuramic acid, L-alanine, and ATP were estimated; 100, 20, and 450 microM, respectively. The specificity of the enzyme for its substrates was investigated with various analogues. Preliminary experiments attempting to elucidate the enzymatic mechanism were consistent with the formation of an acylphosphate intermediate.

Alanine↗