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 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

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

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

A codon-optimized recombinant ribonuclease, MC1 is characterized for its uridine-specific cleavage ability to map nucleoside modifications in RNA. The published MC1 amino acid sequence, as noted in a previous study, was used as a template to construct a synthetic gene with a natural codon bias favoring expression in Escherichia coli. Following optimization of various expression conditions, the active recombinant ribonuclease was successfully purified as a C-terminal His-tag fusion protein from E. coli [Rosetta 2(DE3)] cells. The isolated protein was tested for its ribonuclease activity against oligoribonucleotides and commercially available E. coli tRNA(Tyr I). Analysis of MC1 digestion products by ion-pairing reverse phase liquid-chromatography coupled with mass spectrometry (IP-RP-LC-MS) revealed enzymatic cleavage of RNA at the 5'-termini of uridine and pseudouridine, but cleavage was absent if the uridine was chemically modified or preceded by a nucleoside with a bulky modification. Furthermore, the utility of this enzyme to generate complementary digestion products to other common endonucleases, such as RNase T1, which enables the unambiguous mapping of modified residues in RNA is demonstrated.
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PMID:Detection of RNA nucleoside modifications with the uridine-specific ribonuclease MC1 from Momordica charantia. 2622 Oct 47