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)

The gene for the extracellular ribonuclease of B. pumilus KMM62 (RNase Bp) has been cloned and sequenced. The structural gene for this enzyme is similar to those of the extracellular ribonucleases of B. intermedius 7P (binase) and B. amyloliquefaciens H2 (barnase), as are the regulatory regions of binase and RNase Bp. The regulatory region of the barnase gene, however, is quite different from the other two. In the promoter of the genes for binase and RNase Bp, but not in that for barnase, is a region similar to the Pho box of E. coli. We have established that inorganic phosphate suppresses the synthesis of the binase and RNase Bp, but does not effect the synthesis of barnase.
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PMID:Phosphate regulation of biosynthesis of extracellular RNases of endospore-forming bacteria. 800 70

Ribonucleases catalyze the hydrolysis of the P-O5' bond in RNA. This reaction occurs in two steps: transphosphorylation of RNA to a 2',3'-cyclic phosphodiester intermediate and hydrolysis of this intermediate to a 3'-phosphomonoester. 31P NMR spectroscopy was used to monitor the accumulation of the 2',3'-cyclic phosphodiester intermediate during the transphosphorylation and hydrolysis reactions catalyzed by various ribonucleases and by small molecules. The intermediate was found to accumulate during catalysis by monomeric bovine pancreatic ribonuclease A (RNase A), a dimer and a trimer of RNase A, bovine seminal ribonuclease, RNase T1, barnase, and RNase 1. These enzymes, which are of widely disparate phylogenetic origin, released rather than hydrolyzed most of the intermediate formed transphosphorylation of RNA. In contrast, the intermediate did not accumulate during catalysis by hydroxide ion or imidazole buffer. In the presence of these small molecules, hydrolysis is faster than transphosphorylation. A trapping experiment was used to assess the throughput of the reaction catalyzed by RNase A. [5,6-3H]Uridylyl-(3'-->5')adenosine was incubated with RNase A in the presence of excess unlabeled uridine 2',3'-cyclic phosphodiester, which dilutes the specific radioactivity of any released cyclic intermediate. Only 0.1% of the RNA substrate was found to be both transphosphorylated and hydrolyzed without dissociating from the enzyme. These results suggest that ribonucleases have evolved primarily to catalyze RNA transphosphorylation and not RNA hydrolysis.
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PMID:Energetics of catalysis by ribonucleases: fate of the 2',3'-cyclic phosphodiester intermediate. 800 6

Recombinant ribonuclease (RNase) T1 variants were characterized kinetically taking into account the different reactions catalyzed by this enzyme. In addition to established assays, monitoring the transesterification activity, a photometric assay for fast screening of RNase T1 and variants thereof for ester hydrolysis activity is described, which is based on the application of phenol red as pH indicator. Moreover we established an HPLC assay to evaluate RNase T1 variants by their ability to carry out the transesterification towards an internucleotide diphosphoester (reverse or synthetic activity). In this way we found that the transesterification and hydrolyzing activities of variants change in various directions though in all reactions the same active site and the same transition state are involved. The variant where Tyr42 has been replaced by Trp performs RNA synthesis better than the wild type protein. The scheme of the hypothetic RNase T1 mechanism had to be improved to take into account the non processive character of the reaction.
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PMID:Extended kinetic analysis of ribonuclease T1 variants leads to an improved scheme for the reaction mechanism. 812 15

We have reported previously about the cloning of the binase gene in E. coli. In this work, using an original approach named "homolog gene recombination" method (HGR), vectors for binase expression in E. coli have been constructed. Transcription of the binase gene have been directed through either tac-promoter or PR-promoter of bacteriophage lambda under the control of temperature-sensitive CI857 repressor. The last promoter gave the maximum yield of binase, up to 100 mg of protein per litre of heat-induced bacterial culture. The location of the transcription terminator at the 3' terminus of the binase gene raised the expression approximately two times more. A chromatographic method have been developed and applied for the control of binase accumulation in growth medium without measuring the ribonuclease activity.
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PMID:[Superproduction of Bacillus intermedius 7P ribonuclease (binase) in Escherichia coli]. 818 78

The gene for extracellular low molecular weight ribonuclease of Bacillus circulans BCF 247 was cloned. The strain was isolated from permafrost deposits of the Kolyma lowland. The gene for the ribonuclease from Bacillus intermedius (binase) was used as a specific probe. The cloning succeeded only in the E. coli strain producing the inhibitor of ribonuclease form Bacillus amyloliquefaciens. Selected clones secreted the active ribonuclease into the growth media. Deletion derivatives of the parental recombinant plasmid were constructed. The smallest DNA fragment which enclosed a functional ribonuclease gene in E. coli was determined to be 0.6 kb in length.
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PMID:[Cloning of the gene for extracellular Bacillus circulans RNAase]. 818 79

A ribonuclease T1 homologue, ribonuclease Ms (RNase Ms) from Aspergillus saitoi, has been crystallized as a complex with a substrate analogue GfpC where the 2'-hydroxyl (2'-OH) group of guanosine in guanylyl-3',5'-cytidine (GpC) is replaced by the 2'-fluorine (2'-F) atom to prevent transesterification. The crystal structure of the complex was solved at 1.8-A resolution to a final R-factor of 0.204. The role of His92 (RNase T1 numbering) as the general acid catalyst was confirmed. Of the two alternative candidates for a general base to abstract a proton from the 2'-OH group, His40 and Glu58 were found close to the 2'-F atom, making the decision between the two groups difficult. We then superposed the active site of the RNase Ms/GfpC complex with that of pancreatic ribonuclease S (RNase S) complexed with a substrate analogue UpcA, a phosphonate analogue of uridylyl-3',5'-adenosine (UpA), and found that His12 and His119 of RNase A almost exactly coincided with Glu58 and His92, respectively, of RNase Ms. Similar superposition with a prokaryotic microbial ribonuclease, RNase St [Nakamura, K. T., Iwahashi, K., Yamamoto, Y., Iitaka, Y., Yoshida, N., & Mitsui, Y. (1982) Nature 299, 564-566], also indicated Glu58 as a general base. Thus the present comparative geometrical studies consistently favor, albeit indirectly, the traditional as well as the most recent notion [Steyaert, J., Hallenga, K., Wyns, L., & Stanssens, P. (1990) Biochemistry 29, 9064-9072] that Glu58, rather than His40, must be the general base catalyst in the intact enzymes of the RNase T1 family.
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PMID:Crystal structure of ribonuclease Ms (as a ribonuclease T1 homologue) complexed with a guanylyl-3',5'-cytidine analogue. 821 54

RNase F1, a guanine-specific ribonuclease from Fusarium moniliforme, was crystallized in two different forms, in the absence of an inhibitor and in the presence of 2'GMP. The crystal structure of the RNase F1 free form was solved by the molecular replacement method, using the co-ordinates of the RNase T1 complex with 2'GMP, and was refined to a final R-factor of 18.7%, using the data extended to 1.3 A resolution. For the crystal structure of the RNase F1 complex with 2'GMP, the solution of the molecular replacement method was obtained on the basis of the co-ordinates of the RNase F1 free form, and was refined to a final R-factor of 16.8%, using the data up to 2 A resolution. The two crystal structures of the RNase F1 free form and the complex with 2'GMP are very similar to each other as reflected by a small root-mean-square displacement (r.m.s.d.) value of 0.43 A for all C alpha atoms. The main differences between the two structures are associated with binding of 2'GMP in the substrate recognition site in the loop between Tyr42 and Glu46. A structural comparison between RNase F1 and RNase T1 shows a substantial similarity between all the C alpha atoms, as evidenced by a r.m.s.d. value of 1.4 A. The loop from residues 32 to 38 was strikingly different between these two enzymes, in both its conformation and its hydrogen bonding schemes. The side-chain of a catalytically active residue, His92, is shifted away from the catalytic site in RNase F1 by 1.3 A and 0.85 A with respect to the corresponding positions in the RNase T1 free form and in the RNase T1 complex with 2'GMP, respectively. In the RNase F1 complex, the guanine base of 2'GMP has a syn conformation about the glycosyl bond, and the furanose ring assumes a 3'-exo pucker, which is different from that found in the complex with RNase T1. In the catalytic site of the RNase F1 complex with 2'GMP, one water molecule was observed, which bridges the phosphate oxygen atoms of 2'GMP and the side-chains of the catalytically important residues, His92 and Arg77, through hydrogen bonds. A water molecule occupying the same position was found in the RNase F1 free form. The significance of this water molecule in the hydrolytic reaction is discussed.
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PMID:Crystal structures of ribonuclease F1 of Fusarium moniliforme in its free form and in complex with 2'GMP. 838 73

The structure of the complex of ribonuclease from Streptomyces aureofaciens (RNase Sa) with exo guanosine 2',3'-cyclophosphorothioate has been refined against 0.2-nm resolution synchrotron data using, as a starting model, coordinates from the RNase Sa: 2'-GMP complex. The refinement was based on all data over 1.0-0.2 nm and converged to a crystallographic R factor of 11.9%. This is the first structure of a microbial ribonuclease complexed with a 2',3'-cyclophosphorothioate, which is a thio analogue of the intermediate of the two-step reaction. However, exo guanosine 2',3'-cyclophosphorothioate is bound in a non-functional mode and is not hydrolysed. This structure therefore does not provide direct evidence on the identity of the amino acid residues responsible for catalytic cleavage of the substrate. However, based on present and previous results, a plausible model is proposed for the complex of the cyclic intermediate which acts as substrate for the second step of the catalysis.
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PMID:Complex of ribonuclease Sa with a cyclic nucleotide and a proposed model for the reaction intermediate. 839 32

A genomic DNA encoding ribonuclease (RNase) T1 from Aspergillus oryzae was cloned using a synthetic oligonucleotide probe. The cloned gene (designated rntA) encoded functional RNase T1, since an A. oryzae transformant with multiple copies of the rntA gene showed higher RNase T1 activity (over 200 times) than a transformant with a vector. A cDNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) with primers corresponding to the 5' terminus and 3' terminus of the reading frame of the rntA gene. Nucleotide sequencing analysis of both DNAs found that RNase T1 had a prepro-sequence consisting of 26 amino acids and the rntA gene had only one intron (114 bp) in the region encoding the signal sequence. The A. oryzae transformant with cDNA controlled by the amyB promoter also showed higher activity (over 300 times), indicating that the cloned cDNA encoded functional RNase T1. On the other hand, the Saccharomyces cerevisiae transformant with cDNA controlled by the GAL1 promoter could not grow on a medium containing galactose. These results suggests that A. oryzae may have a protection mechanism from RNase T1.
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PMID:Cloning and nucleotide sequence of the ribonuclease T1 gene (rntA) from Aspergillus oryzae and its expression in Saccharomyces cerevisiae and Aspergillus oryzae. 853 78

The three-dimensional structure of ribonuclease Rh (RNase Rh), a new class of microbial ribonuclease from Rhizopus niveus, has been determined at 2.0 A resolution. The overall structure of RNase Rh is completely different from those of other previously studied RNases, such as RNase A from bovine pancreas and RNase T1 from Aspergillus oryzae. In the structure of RNase Rh, two histidine residues (His46 and His109) and one glutamic acid residue (Glu105), which were predicted to be critical to the activity from the chemical modification and mutagenesis experiments, are found to be located close together, constructing the active site. The indole ring of Trp49 plays an important role in preserving the active site structure by its stacking interactions with the imidazole ring of His 109, and by hydrogen bonding with the carboxyl group of Glu105. There exists a hydrophobic pocket around the active site, which contains the aromatic side-chain of Trp49 and Tyr57. The results of mutagenesis studies suggest that this pocket is the base binding site of the substrate.
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PMID:The crystal structure of ribonuclease Rh from Rhizopus niveus at 2.0 A resolution. 855 22


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