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:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RNA silencing phenomena, known as post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double-stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. Here, we report cloning and expression of the 218 kDa human Dicer, and characterization of its ribonuclease activity and dsRNA-binding properties. The recombinant enzyme generated approximately 21-23 nucleotide products from dsRNA. Processing of the microRNA let-7 precursor by Dicer produced an apparently mature let-7 RNA. Mg(2+) was required for dsRNase activity, but not for dsRNA binding, thereby uncoupling these reaction steps. ATP was dispensable for dsRNase activity in vitro. The Dicer.dsRNA complex formed at high KCl concentrations was catalytically inactive, suggesting that ionic interactions are involved in dsRNA cleavage. The putative dsRNA-binding domain located at the C-terminus of Dicer was demonstrated to bind dsRNA in vitro. Human Dicer expressed in mammalian cells colocalized with calreticulin, a resident protein of the endoplasmic reticulum. Availability of the recombinant Dicer protein will help improve our understanding of RNA silencing and other Dicer-related processes.
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PMID:Ribonuclease activity and RNA binding of recombinant human Dicer. 1241 4

epsilon-Poly-L-lysine (epsilon-PL) is a homo-poly-amino acid characterized by a peptide bond between carboxyl and epsilon-amino groups of L-lysine. Here we report the cell-free synthesis of epsilon-PL by a sensitive radioisotopic epsilon-PL assay system. In vitro epsilon-PL synthesis depended on ATP and was not affected by ribonuclease, kanamycin, or chloramphenicol. epsilon-PL synthesizing activity was detected in the membrane fraction. The reaction product, epsilon-PL, from L-lysine was identified by MALDI-TOF MS and the number of lysine residues of the epsilon-PL products was apparently 11-34. These results suggest that the biosynthesis of epsilon-PL is nonribosomal peptide synthesis and is catalyzed by membrane bound enzyme(s). The enzyme preparation showing the epsilon-PL synthesizing activity also catalyzed lysine-dependent AMP production and an ATP-PPi exchange reaction, suggesting that L-lysine is adenylated in the first step of epsilon-PL biosynthesis.
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PMID:Biosynthesis of epsilon-poly-L-lysine in a cell-free system of Streptomyces albulus. 1462 18

The initial step of the heme biosynthetic pathway in erythroid cells is catalyzed by an erythroid-specific isoform of 5-aminolevulinate synthase-2 (ALAS2). Previously, an alternatively spliced mRNA isoform of ALAS2 was identified although the functional significance of the encoded protein was unknown. We sought to characterize the contribution of this ALAS2 isoform to overall erythroid heme biosynthesis. Here, we report the identification of three novel ALAS2 mRNA splice isoforms in addition to the previously described isoform lacking exon 4-derived sequence. Quantitation of these mRNAs using ribonuclease protection experiments revealed that the isoform without exon 4-derived sequence represents approximately 35-45% of total ALAS2 mRNA while the newly identified transcripts together represent approximately 15%. Despite the significant amounts of these three new transcripts, their features indicate that they are unlikely to substantially contribute to overall mitochondrial ALAS2 activity. In contrast, in vitro studies show that the major splice variant (lacking exon 4-encoded sequence) produces a functional enzyme, albeit with slightly reduced activity and with affinity for the ATP-specific, beta subunit of succinyl CoA synthase, comparable to that of mature ALAS2. It was also established that the first 49 amino acids of the ALAS2 pre-protein are necessary and sufficient for translocation across the mitochondrial inner membrane and that this process is not affected by the absence of exon 4-encoded sequence. We conclude that the major splice isoform of ALAS2 is functional in vivo and could significantly contribute to erythroid heme biosynthesis and hemoglobin formation.
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PMID:The major splice variant of human 5-aminolevulinate synthase-2 contributes significantly to erythroid heme biosynthesis. 1464 93

DNA ligase D (LigD) catalyzes end-healing and end-sealing steps during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal 3'-phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at a duplex primer-template with a short 3'-ribonucleotide tract. The phosphodiesterase, which cleaves a 3'-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3'-PO4 terminus, requires the vicinal 2'-OH of the penultimate ribose. The phosphomonoesterase converts the terminal ribonucleoside 3'-PO4 to a 3'-OH. Here we show that the PE domain has a 3'-phosphatase activity on an all-DNA primer-template, signifying that the phosphomonoesterase reaction does not depend on a 2'-OH. The distinctions between the phosphodiesterase and phosphomonoesterase activities are underscored by the results of alanine-scanning, limited proteolysis, and deletion analysis, which show that the two reactions depend on overlapping but nonidentical ensembles of protein functional groups, including: (i) side chains essential for both ribonuclease and phosphatase activity (His-42, His-48, Asp-50, Arg-52, His-84, and Tyr-88); (ii) side chains important for 3'-phosphatase activity but not for 3' ribonucleoside removal (Arg-14, Asp-15, Glu-21, Gln-40, and Glu-82); and (iii) side chains required selectively for the 3'-ribonuclease (Lys-66 and Arg-76). These constellations of critical residues are unique to LigD-like proteins, which we propose comprise a new bifunctional phosphoesterase family.
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PMID:Essential constituents of the 3'-phosphoesterase domain of bacterial DNA ligase D, a nonhomologous end-joining enzyme. 1604 7

Mitochondrial aconitase (mACON) contains a [4Fe-4S] cluster as the key enzyme for citrate oxidation in the human prostatic epithelial cell. Although there is accumulating evidence indicating that accumulation of high levels of zinc in prostate epithelial cells causes reduced efficiency of citrate oxidation, zinc regulation on the mACON is still not well understood. From in vitro studies, zinc chloride treatment has been developed using humic acid as the carrier (Zn-HA) in human prostatic carcinoma cells, PC-3. Zn-HA treatment (0.1-10 microM) restricts mACON enzymatic activity, which attenuates citrate utility and decreases intracellular ATP levels in PC-3 cells, whereas the effect is blocked by adding the zinc chelator, diethylenetriaminepentaacetic acid (DTPA). Immunoblot, ribonuclease-protection and transient gene-expression assays indicate that Zn-HA treatments inhibit mACON gene expression. Mutation of the putative metal response element (MRE) from CTCGCCTTCA to TGATCCTTCA abolishes Zn-HA inhibition of mACON promoter activity. Our results have demonstrated that zinc possesses a specific regulatory mechanism on the mACON gene, and a biologic function of the putative metal regulatory system in mACON gene transcription has been identified.
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PMID:Zinc blocks gene expression of mitochondrial aconitase in human prostatic carcinoma cells. 1609 33

ER-60 is a PDI family protein that has protein thiol-disulfide oxidoreductase activity. It has been shown to associate with BiP in the endoplasmic reticulum. Here, we analyzed the cooperation of ER-60 and BiP in the oxidative refolding of denatured proteins in vitro. ER-60 facilitated the refolding of 20 or 30% of denatured alpha-lactalbumin or ribonuclease B during incubation for 80 min, and these levels of nearly doubled on the addition of BiP to the reaction mixture. BiP alone could not refold denatured ribonuclease B, but could refold denatured alpha-lactalbumin a little. Enhancement of oxidative refolding of alpha-lactalbumin by ER-60 could be detected only when ER-60 was present from the start of refolding. On surface plasmon resonance analysis, ER-60 was shown to associate with BiP. The association was not influenced by ATP or ADP. Domains a and/or b' of ER-60 were implicated in the association with BiP.
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PMID:Cooperation of ER-60 and BiP in the oxidative refolding of denatured proteins in vitro. 1642 6

DNA ligase D (LigD) performs end remodeling and end sealing reactions during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at the 3' end of the primer strand of a primer-template. The phosphodiesterase cleaves a 3'-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3'-PO4 terminus. The phosphomonoesterase converts a terminal ribonucleoside 3'-PO4 or deoxyribonucleoside 3'-PO4 of a primer-template to a 3'-OH. Here we report that the phosphodiesterase and phosphomonoesterase activities are both dependent on the presence and length of the 5' single-strand tail of the primer-template substrate. Although the phosphodiesterase activity is strictly dependent on the 2'-OH of the penultimate ribose, it is indifferent to a 2'-OH versus a2'-H on the terminal nucleoside. Incision at the ribonucleotide linkage is suppressed when the 2'-OH is moved by 1 nucleotide in the 5' direction, suggesting that LigD is an exoribonuclease that cleaves the 3'-terminal phosphodiester. We report the effects of conservative amino acid substitutions at residues: (i) His42, His48, Asp50, Arg52, His84, and Tyr88, which are essential for both the ribonuclease and 3'-phosphatase activities; (ii) Arg14, Asp15, Glu21, and Glu82, which are critical for 3'-phosphatase activity but not 3'-ribonucleoside removal; and (iii) at Lys66 and Arg76, which participate selectively in the 3'-ribonuclease reaction. The results suggest roles for individual functional groups in metal binding and/or phosphoesterase chemistry.
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PMID:Substrate specificity and structure-function analysis of the 3'-phosphoesterase component of the bacterial NHEJ protein, DNA ligase D. 1654 Apr 77

The precursor to the light-harvesting chlorophyll a/b protein of photosystem II can insert into isolated thylakoid membranes if reaction mixtures also contain ATP and a soluble extract of chloroplasts. Optimization of this insertion process and the initial characterization of the soluble chloroplastic component are presented. With a fixed amount of precursor, maximum integration rates occurred during the first 30 minutes at pH 8.0 and 30 degrees C when the soluble chloroplast extract was increased eight-fold over the stoichiometric amount. Under these conditions, insertion was routinely about 60% of that which occurred during import into intact chloroplasts. Integration also increased virtually linearly with increasing amounts of precursor. However, assays revealed that at least 40% of the in vitro-synthesized pLHCP was pelletable and inactive. The soluble chloroplastic component exhibited characteristics expected of a protein. It was inactivated by heat, protease, and N-ethylmaleimide, but was insensitive to ribonuclease. The soluble component migrated on a Sephacryl S-200 gel filtration column as a single peak with an M(r) of approximately 65,000. The proteinaceous nature of this factor suggests a similarity to soluble factors required for protein transport/integration in other membrane systems.
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PMID:A Soluble Protein Factor is Required in Vitro for Membrane Insertion of the Thylakoid Precursor Protein, pLHCP. 1666 35

5-Aminolevulinic acid synthesis in isolated, intact, developing chloroplasts from greening cucumber (Cucumis sativus) cotyledons was inhibited by broken chloroplast fragments. It was shown that the inhibitory constituent was associated with the thylakoid membrane system. The inhibitor was resistant to boiling, was not a form of ribonuclease, and did not inhibit Mg-chelatase, indicating that massive organelle destruction was not involved. The inhibitor was also found in etioplast and mature chloroplasts; and it was found in barley as well as cucumber. 5-Aminolevulinate synthesis in the dark with exogenous ATP and NADPH, or in the light without added cofactors, were inhibited approximately equally. In the dark, 5-aminolevulinate synthesis and protochlorophyllide synthesis from glutamate were inhibited to about equal extent. The inhibition was decreased when the membranes were washed with aqueous acetone prior to incubation. The inhibition by the unknown factor was compared to the inhibition by gabaculine, 4-amino-5-hexynoic acid, protoheme, and glutathione. The unknown inhibitor appeared to have a number of similarities with protoheme.
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PMID:Regulation of 5-Aminolevulinic Acid Synthesis in Developing Chloroplasts : IV. An Endogenous Inhibitor from the Thylakoid Membranes. 1666 54

Polyphosphate (polyP) is a linear polymer consisting of tens to hundreds of phosphate molecules joined together by high-energy anhydride bonds. These polymers are found in virtually all prokaryotic and eukaryotic cells and perform many functions; prominent among them are the responses to many stresses. Polyphosphate is synthesized by polyP kinase (PPK), using the terminal phosphate of ATP as the substrate, and degraded to inorganic phosphate by both endo- and exopolyphosphatases. Here we report the crystal structure and analysis of the polyphosphate phosphatase PPX from Escherichia coli O157:H7 refined at 2.2 Angstroms resolution. PPX is made of four domains. Domains I and II display structural similarity with one another and share the ribonuclease-H-like fold. Domain III bears structural similarity to the N-terminal, HD domain of SpoT. Domain IV, the smallest domain, has structural counterparts in cold-shock associated RNA-binding proteins but is of unknown function in PPX. The putative PPX active site is located at the interface between domains I and II. In the crystal structure of PPX these two domains are close together and represent the "closed" state. Comparison with the crystal structure of PPX/GPPA from Aquifex aeolicus reveals close structural similarity between domains I and II of the two enzymes, with the PPX/GPPA representing an "open" state. A striking feature of the dimer is a deep S-shaped canyon extending along the dimer interface and lined with positively charged residues. The active site region opens to this canyon. We postulate that this is a likely site of polyP binding.
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PMID:The structure of the exopolyphosphatase (PPX) from Escherichia coli O157:H7 suggests a binding mode for long polyphosphate chains. 1667 53


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