Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Isolates (n = 94) of Corynebacterium pseudotuberculosis were obtained from sheep, goats, horses, and cattle from various parts of the world. The isolates were characterized biochemically and by restriction endonuclease analysis of DNA. We found near homogeneity in the ability of isolates to ferment carbohydrates and to produce urease. All isolates produced phospholipase D and catalase. The ability of isolates from horses to reduce nitrate, the inability of isolates from sheep and goats to do so, and the correlation of this characteristic with results of restriction endonuclease analyses confirmed the existence of 2 biovars of C pseudotuberculosis. We propose that these biovars be referred to as biovar equi for isolates that reduce nitrate and biovar ovis for isolates that fail to do so.
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PMID:Biochemical and genetic characterization of Corynebacterium pseudotuberculosis. 283 63

Phosphatidylcholine-specific phospholipase D (PLD) enzymes catalyze hydrolysis of phospholipid phosphodiester bonds, and also transphosphatidylation of phospholipids to acceptor alcohols. Bacterial and plant PLD enzymes have not been shown previously to be homologues or to be homologous to any other protein. Here we show, using sequence analysis methods, that bacterial and plant PLDs show significant sequence similarities both to each other, and to two other classes of phospholipid-specific enzymes, bacterial cardiolipin synthases, and eukaryotic and bacterial phosphatidylserine synthases, indicating that these enzymes form an homologous family. This family is suggested also to include two Poxviridae proteins of unknown function (p37K and protein K4), a bacterial endonuclease (nuc), an Escherichia coli putative protein (o338) containing an N-terminal domain showing similarities with helicase motifs V and VI, and a Synechocystis sp. putative protein with a C-terminal domain likely to possess a DNA-binding function. Surprisingly, four regions of sequence similarity that occur once in nuc and o338, appear twice in all other homologues, indicating that the latter molecules are bi-lobed, having evolved from an ancestor or ancestors that underwent a gene duplication and fusion event. It is suggested that, for each of these enzymes, conserved histidine, lysine, aspartic acid, and/or asparagine residues may be involved in a two-step ping pong mechanism involving an enzyme-substrate intermediate.
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PMID:A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases: identification of duplicated repeats and potential active site residues. 873 63

A family of phospholipase D (PLD) proteins has recently been identified (Koonin, 1996; Ponting & Kerr, 1996) based upon amino acid sequence identity. This family includes human and plant PLDs, proteins encoded by open reading frames in pathogenic viruses and bacteria, as well as an endonuclease. The endonuclease, known as Nuc, is encoded by the IncN plasmid, pKM101, present in Salmonella typhimurium. The recombinant Nuc protein has been expressed and purified from Escherichia coli. The amino-terminal sequencing of the purified protein indicated that the mature protein started from the 23rd residue of the predicted sequence, suggesting that the protein is proteolytically processed during export to the periplasmic space. The recombinant enzyme was able to hydrolyze both double and single-strand DNA and an artificial substrate, bis(4-nitrophenyl) phosphate, which contains a phosphodiester bond. The enzyme activity was not inhibited in the presence of EDTA and was not regulated by divalent cations. The purified protein has been crystallized by hanging drop vapor diffusion methods, and those crystals diffract to 1.9 A resolution.
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PMID:Expression, characterization, and crystallization of a member of the novel phospholipase D family of phosphodiesterases. 941 18

The phospholipase D (PLD) superfamily includes enzymes of phospholipid metabolism, nucleases, as well as ORFs of unknown function in viruses and pathogenic bacteria. These enzymes are characterized by the invariant sequence motif, H(X)K(X)4D. The endonuclease member Nuc of the PLD family was over-expressed in bacteria and purified to homogeneity. Mutation of the conserved histidine to an asparagine in the endonuclease reduced the kcat for hydrolysis by a factor of 10(5), suggesting that the histidine residue plays a key role in catalysis. In addition to catalyzing hydrolysis, a number of phosphohydrolases will catalyze a phosphate (oxygen)-water exchange reaction. We have taken advantage of this observation and demonstrate that a 32P-labeled protein could be trapped when the enzyme was incubated with 32P-labeled inorganic phosphate. The phosphoenzyme intermediate was stable in 1 M NaOH and labile in 1 M HCl and 1 M hydroxylamine, suggesting that the enzyme forms a phosphohistidine intermediate. The pH-stability profile of the phosphoenzyme intermediate was consistent with phosphohistidine and the only radioactive amino acid found after alkaline hydrolysis was phosphohistidine. These results suggest that the enzymes in the PLD superfamily use the conserved histidine for nucleophilic attack on the substrate phosphorus atom and most likely proceed via a common two-step catalytic mechanism.
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PMID:Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate. 968 58

BfiI is a novel type IIs restriction endonuclease that, unlike all other restriction enzymes characterised to date, cleaves DNA in the absence of Mg(2+). The amino acid sequence of the N-terminal part of BfiI has some similarities to Nuc of Salmonella typhimurium, an EDTA-resistant nuclease akin to phospholipase D. The dimeric form of Nuc contains a single active site composed of residues from both subunits. To examine the roles of the amino acid residues of BfiI that align with the catalytic residues in Nuc, a set of alanine replacement mutants was generated by site-directed mutagenesis. The mutationally altered forms of BfiI were all catalytically inactive but were still able to bind DNA specifically. The active site of BfiI is thus likely to be similar to that of Nuc. BfiI was also found by gel-filtration to be a dimer in solution. Both gel-shift and pull-down assays indicated that the dimeric form of BfiI binds two copies of its recognition sequence. In reactions on plasmids with either one or two copies of its recognition sequence, BfiI cleaved the DNA with two sites more rapidly than that with one site. Yet, when bound to two copies of its recognition sequence, the BfiI dimer cleaved only one phosphodiester bond at a time. The dimer thus seems to contain two DNA-binding domains but only one active site.
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PMID:The metal-independent type IIs restriction enzyme BfiI is a dimer that binds two DNA sites but has only one catalytic centre. 1258 53

The role of bivalent cations and choline in ATP-induced apoptosis via P2Z purinoceptor was investigated in human leukemic lymphocytes. In vitro exposure of leukemic lymphocytes with P2Z receptors to 1 mmol/L ATP or 0.1 mmol/L benzoylbenzoic ATP (BzATP) for 8 h in the presence of choline, 1 mmol/L Mg2+ or other bivalent cations, and ATP-induced DNA breaks, associated with apoptosis were quantified by TdT assay. We observed that (1) Extracellular Mg2+ or Ca2+ stimulated ATP-induced DNA fragmentation in a dose-dependent manner, and the compatible evidence was provided by the inhibition of ATP-induced DNA fragmentation in the present of EGTA or EDTA; (2) ATP-induced DNA fragmentation was completely inhibited by 1 mmol/L Zn2+; (3) ATP-induced DNA breaks were not affected by Ba2+, Sr2+, Co2+ when they were substituted for extracellular Mg2+ or Ca2+; (4) Choline, an inhibitor of phospholipase D (PLD) stimulated by ATP through P2Z receptor in human lymphocytes, was also a partial inhibitor of ATP-induced DNA fragmentation, and the results were confirmed by flow cytometric analysis (FCA); (5) ATP-induced DNA fragmentation was completely obliterated when the temperature was lower than 10 degrees C. These results suggest that the endonuclease and PLD may be involved in ATP-induced apoptosis in human lymphocytes via P2Z receptor.
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PMID:Role of bivalent cations and choline in ATP-induced apoptosis of human lymphocytes with P2Z receptors. 1289 80

The BfiI endonuclease cleaves DNA at fixed positions downstream of an asymmetric sequence. Unlike other restriction enzymes, it functions without metal ions. The N-terminal half of BfiI is similar to Nuc, an EDTA-resistant nuclease from Salmonella typhimurium that belongs to the phosphoplipase D superfamily. Nuc is a dimer with one active site at its subunit interface, as is BfiI, but it cuts DNA non-specifically. BfiI was cleaved by thermolysin into an N-terminal domain, which forms a dimer with non-specific nuclease activity, and a C-terminal domain, which lacks catalytic activity but binds specifically to the recognition sequence as a monomer. On denaturation with guanidinium, BfiI underwent two unfolding transitions: one at a relatively low concentration of guanidinium, to a dimeric non-specific nuclease; a second at a higher concentration, to an inactive monomer. The isolated C-terminal domain unfolded at the first (relatively low) concentration, the isolated N-terminal at the second. Hence, BfiI consists of two physically separate domains, with catalytic and dimerisation functions in the N terminus and DNA recognition functions in the C terminus. It is the first example of a restriction enzyme generated by the evolutionary fusion of a DNA recognition domain to a phosphodiesterase from the phospholipase D superfamily. BfiI may consist of three structural units: a stable central core with the active site, made from two copies of the N-terminal domain, flanked by relatively unstable C-terminal domains, that each bind a copy of the recognition sequence.
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PMID:Generation of the BfiI restriction endonuclease from the fusion of a DNA recognition domain to a non-specific nuclease from the phospholipase D superfamily. 1474 Dec 5

To improve the control of Chlamydia trachomatis infection in India, a rapid, specific and cost-effective method is much needed. We developed an in-house PCR assay by targeting a unique genomic sequence encoding a protein from the C. trachomatis phospholipase D endonuclease superfamily that produces an amplified fragment of 368 bp. The specificity of the primers was confirmed using genomic DNA from other sexually transmitted disease-causing and related micro-organisms and from humans. The assay was highly sensitive and could detect as low as 10 fg C. trachomatis DNA. Clinical evaluation of the in-house-developed PCR was carried out using 450 endocervical specimens that were divided in two groups. In group I (n=274), in-house PCR was evaluated against the direct fluorescence assay. The resolved sensitivity of the in-house PCR method was 97.22 % compared with 88 % for the direct fluorescent antibody assay. In group II (n=176), the in-house PCR was compared with the commercial Roche AMPLICOR MWP CT detection kit. The resolved sensitivity of the in-house PCR assay reported here was 93.1 % and the specificity was 97.46 %, making it a cost-effective alternative for routine diagnosis of genital infection by C. trachomatis. The method should facilitate early detection leading to better prevention and treatment of genital infection in India.
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PMID:Comparison of an in-house PCR assay, direct fluorescence assay and the Roche AMPLICOR Chlamydia trachomatis kit for detection of C. trachomatis. 1950 71

Metal-dependent nucleases that generate double-strand breaks in DNA often possess two symmetrically-equivalent subunits, arranged so that the active sites from each subunit act on opposite DNA strands. Restriction endonuclease BfiI belongs to the phospholipase D (PLD) superfamily and does not require metal ions for DNA cleavage. It exists as a dimer but has at its subunit interface a single active site that acts sequentially on both DNA strands. The active site contains two identical histidines related by 2-fold symmetry, one from each subunit. This symmetrical arrangement raises two questions: first, what is the role and the contribution to catalysis of each His residue; secondly, how does a nuclease with a single active site cut two DNA strands of opposite polarities to generate a double-strand break. In this study, the roles of active-site histidines in catalysis were dissected by analysing heterodimeric variants of BfiI lacking the histidine in one subunit. These variants revealed a novel mechanism for the scission of double-stranded DNA, one that requires a single active site to not only switch between strands but also to switch its orientation on the DNA.
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PMID:A novel mechanism for the scission of double-stranded DNA: BfiI cuts both 3'-5' and 5'-3' strands by rotating a single active site. 2004 64

The genomes of two Bacillus cereus strains (ATCC 10987 and ATCC 14579) have been sequenced. Here, we report the specificities of type II/III restriction (R) and modification (M) enzymes. Found in the ATCC 10987 strain, BceSI is a restriction endonuclease (REase) with the recognition and cut site CGAAG 24-25/27-28. BceSII is an isoschizomer of AvaII (G/GWCC). BceSIII cleaves at ACGGC 12/14. The BceSIII C terminus resembles the catalytic domains of AlwI, MlyI, and Nt.BstNBI. BceSIV is composed of two subunits and cleaves on both sides of GCWGC. BceSIV activity is strongly stimulated by the addition of cofactor ATP or GTP. The large subunit (R1) of BceSIV contains conserved motifs of NTPases and DNA helicases. The R1 subunit has no endonuclease activity by itself; it strongly stimulates REase activity when in complex with the R2 subunit. BceSIV was demonstrated to hydrolyze GTP and ATP in vitro. BceSIV is similar to CglI (GCSGC), and homologs of R1 are found in 11 sequenced bacterial genomes, where they are paired with specificity subunits. In addition, homologs of the BceSIV R1-R2 fusion are found in many sequenced microbial genomes. An orphan methylase, M.BceSV, was found to modify GCNGC, GGCC, CCGG, GGNNCC, and GCGC sites. A ParB-methylase fusion protein appears to nick DNA nonspecifically. The ATCC 14579 genome encodes an active enzyme Bce14579I (GCWGC). BceSIV and Bce14579I belong to the phospholipase D (PLD) family of endonucleases that are widely distributed among Bacteria and Archaea. A survey of type II and III restriction-modification (R-M) system genes is presented from sequenced B. cereus, Bacillus anthracis, and Bacillus thuringiensis strains.
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PMID:Characterization of type II and III restriction-modification systems from Bacillus cereus strains ATCC 10987 and ATCC 14579. 2203 2


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