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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two ribonucleases (RNase Phya and RNase Phyb) were purified to homogeneity on SDS-PAGE from the culture filtrate of the fungus Physarum polycephalum. The apparent molecular weights of RNases Phya and Phyb were about 20,000. The pH optima of these two RNases were around 4.5-4.75. The RNases released mononucleotides from RNA in the order of 3'-GMP, 3'-AMP, and 3'-pyrimidine nucleotides. RNase Phya and RNase Phyb have the N-terminal amino acid sequences STSFD--- and KSTSF--, respectively. This finding and the similar amino acid compositions of both RNases indicated that they might share the same protein moiety except for the N-terminus Lys. The complete primary structure of RNase Phyb was determined, mostly by analysis of the peptides generated by trypsin, V8 protease, and lysylendopeptidase digestions. The molecular weight of the protein moiety was 19,704. The locations of four half cystine residues were almost superimposable on those in five known fungal RNase T2 family RNases, but two others were not. The sequence homology between RNase Phyb and five known fungal RNases amounted to 53-59 residues, which are concentrated around the three histidine residues, supposed to form the active site in enzymes of the RNase T2 family. However, the amino acid sequence of RNase Phyb more closely resembles those of plant RNases such as RNases from Nicotiana alata [McClure, B.A. et al. (1989) Nature 342, 955-957], tomato [RNase Le, Yost et al. (1991) Eur. J. Biochem. 198, 1-6], and Momoridica charantia [RNase MC1, Ide et al. (1991) FEBS Lett. 284, 161-164].
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PMID:Purification, some properties, and primary structure of base non-specific ribonucleases from Physarum polycephalum. 851 32

The ribonuclease MC1 (RNase MC1) from seeds of bitter gourd (Momordica charantia) consists of 190 amino acid residues with four disulfide bridges and belongs to the RNase T(2) family, including fungal RNases typified by RNase Rh from Rhizopus niveus and RNase T(2) from Aspergillus oryzae. The crystal structure of RNase MC1 has been determined at 1.75 A resolution with an R-factor of 19.7% using the single isomorphous replacement method. RNase MC1 structurally belongs to the (alpha+beta) class of proteins, having ten helices (six alpha-helices and four 3(10)-helices) and eight beta-strands. When the structures of RNase MC1 and RNase Rh are superposed, the close agreement between the alpha-carbon positions for the total structure is obvious: the root mean square deviations calculated only for structurally related 151 alpha-carbon atoms of RNase MC1 and RNase Rh molecules was 1.76 A. Furthermore, the conformation of the catalytic residues His-46, Glu-105, and His-109 in RNase Rh can be easily superposed with that of the possible catalytic residues His-34, Glu-84, and His-88 in RNase MC1. This observation strongly indicates that RNase MC1 from a plant origin catalyzes RNA degradation in a similar manner as fungal RNases.
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PMID:Crystal structure of a ribonuclease from the seeds of bitter gourd (Momordica charantia) at 1.75 A resolution. 1044 75

The ribonuclease MC1 (RNase MC1) from seeds of bitter gourd (Momordica charantia) consists of 190 amino acids and belongs to the RNase T2 family, including fungal RNases typified by RNase Rh from Rhizopus niveus. We expressed RNase MC1 in Escherichia coli cells and made use of site-directed mutagenesis to identify essential amino acid residues for catalytic activity. Mutations of His34 and His88 to Ala completely abolished the enzymatic activity, and considerable decreases in the enzymatic activity were observed in cases of mutations of His83, Glu84, and Lys87, when yeast RNA was used as a substrate. Kinetic parameters for the enzymatic activity of the mutants of His83, Glu84, and Lys87 were analyzed using a dinucleoside monophosphate CpU. Km values for the mutants were approximately like that for wild-type, while k(cat) values were decreased by about 6 to 25-fold. These results suggest that His34, His83, Glu84, Lys87, and His88 in RNase MC1 may be involved in the catalytic function. These observation suggests that RNase MC1 from a plant catalyzes RNA degradation in a similar manner to that of fungal RNases.
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PMID:Expression and mutational analysis of amino acid residues involved in catalytic activity in a ribonuclease MC1 from the seeds of bitter gourd. 1080 62

Ribonuclease MC1 (RNase MC1) isolated from seeds of bitter gourd (Momordica charantia) consists of 190 amino acids and is characterized by a preferential cleavage at the 5'-side of uridine. This uridine specificity distinguishes RNase MC1 from other enzymes belonging to the RNase T2 family. The three-dimensional structures of RNase MC1, in a complex with either 2'-UMP or 3'-UMP, were determined at 1.48 and 1.77 A resolutions, respectively. The side chains of Gln9 and Asn71 interact with O4 and N3, respectively, of the uracil base by hydrogen bondings. In addition, the uracil base is sandwiched by the hydrophobic side chains of Leu73 and Phe80. Compared with these amino acid residues and corresponding residues in RNases in the RNase T2 family, Gln9 and Phe80 are highly conserved in the RNases in T2 family, while Asn71 and Leu73 in RNase MC1 are variant in sequences. It is thus likely that interactions of the side chains of Asn71 and Leu73 with the uracil base are responsible for the absolute uridine specificity of RNase MC1. Site-directed mutagenesis experiments showed that replacement of Asn by Thr decreased both the catalytic efficiency and the binding affinity by 2.3- and 7.0-fold, respectively, and substitution of Leu73 for Ala predominantly decreased the binding affinity by 14. 5-fold, compared with findings in case of wild-type RNase MC1. It is thus demonstrated that Asn71 and Leu73 play an essential role in uridine preference for RNase MC1.
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PMID:Crystal structures of the ribonuclease MC1 from bitter gourd seeds, complexed with 2'-UMP or 3'-UMP, reveal structural basis for uridine specificity. 1096 5

The ribonuclease MC1 (RNase MC1), isolated from seeds of bitter gourd (Momordica charantia), consists of 190 amino acids and is characterized by specific cleavage at the 5'-side of uridine. Site-directed mutagenesis was used to evaluate the contribution of four amino acids, Asn71, Val72, Leu73, and Arg74, at the alpha4-alpha5 loop between alpha4 and alpha5 helices for recognition of uracil base by RNase MC1. Four mutants, N71T, V72L, L73A, and R74S, in which Asn71, Val72, Leu73, and Arg74 in RNase MC1 were substituted for the corresponding amino acids, Thr, Leu, Ala, and Ser, respectively, in a guanylic acid preferential RNase NW from Nicotiana glutinosa, were prepared and characterized with respect to enzymatic activity. Kinetic analysis with a dinucleoside monophosphate, CpU, showed that the mutant N71T exhibited 7.0-fold increased K(m) and 2.3-fold decreased k(cat), while the mutant L73A had 14.4-fold increased K(m), although it did retain the k(cat) value comparable to that of the wild-type. In contrast, replacements of Val72 and Arg74 by the corresponding amino acids Leu and Ser, respectively, had little effect on the enzymatic activity. This observation is consistent with findings in the crystal structure analysis that Asn71 and Leu73 are responsible for a uridine specificity for RNase MC1. The role of Asn71 in enzymatic reaction of RNase MC1 was further investigated by substituting amino acids Ala, Ser, Gln, and Asp. Our observations suggest that Asn71 has at least two roles: one is base recognition by hydrogen bonding, and the other is to stabilize the conformation of the alpha4-alpha5 loop by hydrogen bonding to the peptide backbone, events which possibly result in an appropriate orientation of the alpha-helix (alpha5) containing active site residues. Mutants N71T and N71S showed a remarkable shift from uracil to guanine specificity, as evaluated by cleavage of CpG, although they did exhibit uridine specificity against yeast RNA and homopolynucleotides.
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PMID:Amino acid residues in ribonuclease MC1 from bitter gourd seeds which are essential for uridine specificity. 1114 47

Ribonuclease MC1 (RNase MC1) isolated from bitter gourd (Momordica charantia) seeds specifically cleaves phosphodiester bonds on the 5'-side of uridine. The crystal structures of RNase MC1 in complex with 2'-UMP or 3'-UMP reveal that Gln9, Asn71, Leu73, and Phe80 are involved in uridine binding by hydrogen bonding and hydrophobic interactions [Suzuki et al. (2000) Biochem. Biophys. Res. Commun. 275, 572-576]. To evaluate the contribution of Gln9 and Phe80 to uridine binding, Gln9 was replaced with Ala, Phe, Glu, or His, and Phe80 with Ala by site-directed mutagenesis. The kinetic properties of the resulting mutant enzymes were characterized using cytidylyl-3',5'-uridine (CpU) as a substrate. The mutant Q9A exhibited a 3.7-fold increased K(m) and 27.6-fold decreased k(cat), while three other mutations, Q9F, Q9E, and Q9H, predominantly affected the k(cat) value. Replacing Phe80 with Ala drastically reduced the catalytic efficiency (k(cat)/K(m)) with a minimum K(m) value equal to 8 mM. It was further found that the hydrolytic activities of the mutants toward cytidine-2',3'-cyclic monophosphate (cCMP) were reduced. These results demonstrate that Gln9 and Phe80 play essential roles not only in uridine binding but also in hydrolytic activity. Moreover, we produced double Ala substituted mutants at Gln9, Asn71, Leu73, and Phe80, and compared their kinetic properties with those of the corresponding single mutants. The results suggest that these four residues may contribute to uridine binding in a mutually independent manner.
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PMID:Contribution of Gln9 and Phe80 to substrate binding in ribonuclease MC1 from bitter gourd seeds. 1168 24

Ribonuclease NW (RNase NW), the wound-inducible RNase in Nicotiana glutinosa leaves, preferentially cleaves guanylic acid. We expressed the cDNA encoding RNase NW in the methylotrophic yeast Pichia pastoris using the expression vector pPIC9K, and the resulting recombinant RNase NW (ryRNaseNW) secreted into medium was purified to apparent homogeneity using column chromatography. The crystal structure of ryRNase NW bound to 5'-GMP was determined at 1.5 A resolution by molecular replacement with tomato RNase LE as a search model. The RNase NW structurally belongs to the (alpha + beta) class of proteins, having eight helices (five alpha-helices and three 3(10) helices) and six beta-strands, and its structure is highly similar to those of other plant RNases, including a uridylic acid preferential RNase MC1 from bitter gourd seeds. The guanine ring of 5'-GMP lies in a hydrophobic pocket of the molecular surface composed of Tyr17, Tyr71, Ala80, Leu79, and Phe89: the guanine base is sandwiched between aromatic side chains of Tyr17 and Phe89. In addition, the guanine base is firmly stabilized by a network of hydrogen bonds of the side chains of Gln12 and Thr78, as well as of the main chain of Leu79. Therefore, Gln12, Tyr17, Thr78, Leu79, and Phe89 are responsible for recognition of the guanine base by RNase NW, findings which provide insight into the manner in which RNase NW preferentially cleaves guanylic acid.
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PMID:Guanine binding site of the Nicotiana glutinosa ribonuclease NW revealed by X-ray crystallography. 1248 57

Ribonuclease MC1 (RNase MC1), isolated from bitter gourd seeds, is a uridine specific RNase belonging to the RNase T2 family. Mutations of Asn71 in RNase MC1 to the amino acids Thr (N71T) and Ser (N71S) in guanosine preferential RNases altered the substrate specificity from uridine specific to guanosine specific, as shown by the transphosphorylation of diribonucleoside monophosphates [Numata, T., et al. (2001) Biochemistry 40, 524-530]. To elucidate the structural basis for the alteration of substrate specificity, crystal structures of the RNase MC1 mutants N71T and N71S, free or complexed with 5'-GMP, were determined at resolutions higher than 2 A. In the N71T-5'-GMP and N71S-5'-GMP complexes, the guanine moiety was, as in the case of the uracil moiety bound to wild-type RNase MC1, firmly stabilized in the B2 site by an extensive network of hydrogen bonds and hydrophobic interactions. Structure comparisons showed that mutations of Asn71 to Thr or Ser cause an enlargement of the B2 site, which then make it feasible to insert a guanine base into the B2 site of mutants N71T and N71S. This binding further allows for hydrogen bonding interaction of the side chain hydroxyl groups of Thr71 or Ser71 with the N7 atom of the guanine base. The mode of guanine binding of mutants N71T and N71S was found to be essentially identical to that of a guanosine preferential RNase NW from Nicotiana glutinosa. In particular, hydrogen bonds between the N7 atom of the guanine base and the hydroxyl groups of the amino acids at position 71 (RNase MC1 numbering) were completely conserved in three guanosine preferential enzymes, thereby indicating that the hydrogen bond may play an essential role in guanine binding in guanosine preferential RNases in the RNase T2 family. Consequently, it can be concluded that amino acids at position 71 (RNase MC1 numbering) serve as one of the determinants for substrate specificity (or preference) in the RNase T2 fimily by changing the size and shape of the B2 site.
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PMID:Crystal structures of the ribonuclease MC1 mutants N71T and N71S in complex with 5'-GMP: structural basis for alterations in substrate specificity. 1273 68

The ribonuclease MC1 (RNase MC1) from the seeds of the bitter gourd belongs to the RNase T2 family. We evaluated the contribution of 11 amino acids conserved in the RNase T2 family to protein folding of RNase MC1. Thermal unfolding experiments showed that substitution of Tyr(101), Phe(102), Ala(105), and Phe(190) resulted in a significant decrease in themostability; the T(m) values were 47-58 degrees C compared to that for the wild type (64 degrees C). Mutations of Pro(125), Gly(127), Gly(144), and Val(165) caused a moderate decrease in thermostability (T(m): 60-62 degrees C). In contrast, mutations of Asp(107) and Gly(173) did little effect on thermostability. The contribution of Tyr(101), Phe(102), Pro(125), and Gly(127) to protein stability was further corroborated by means of Gdn-HCl unfolding and protease digestions. Taken together, it appeared that Tyr(101), Phe(102), Ala(105), Pro(125), Gly(127), Gly(144), Leu(162), Val(165), and Phe(190) conserved in the RNase T2 family play an important role in the stability of the proteins.
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PMID:Amino acids conserved at the C-terminal half of the ribonuclease T2 family contribute to protein stability of the enzymes. 1532 60

A base non-specific ribonuclease (RNase Bm2) was isolated from a green algae (Ulvophyceae, Bryopsis maxima) as a single band on SDS-PAGE, and its primary structure and enzymatic properties, including base specificity, were investigated. The amino acid sequence of RNase Bm2 was homologous to many RNase T2 family RNases, and their characteristic CAS sequences were also conserved. The molecular mass of RNase Bm2 was 24444 Da, and its optimal pH was 5.5. RNase Bm2 was a poly U preferential RNase, similar to RNase MC1 from bitter gourd. The base specificity of this RNase suggested that the base specificity of the B1- and B2-base binding sites of RNase Bm2 were G > or = U > C >> A and U > G > C >> A, respectively. The estimated active site of RNase Bm2 was very similar to that of RNase MC1 from bitter gourds; however, a tyrosine residue at the B1-base binding site that is conserved for all RNase T2 family RNases was replaced by a tryptophan residue. Here we discuss the effect of this replacement on the base specificity of RNase Bm2 and the phylogenetic relationship of RNase T2 family enzymes.
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PMID:Primary structure and properties of ribonuclease Bm2 (RNase Bm2) from Bryopsis maxima. 1665 12


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