title: Enzymatic properties of mutant forms of RNase Rh from Rhizopus niveus as to Asp51.
abstract: In order to determine the role of Asp51 of RNase Rh from Rhizopus niveus, enzymes with mutations at the 51st position, D51N, D51E, D51Q, D51S, D51T, D51A, and D51K, were prepared, and their enzymatic properties were investigated as to specific activity and base specificity. All the mutant enzymes showed relatively high activity toward poly I and poly C, and markedly reduced activity toward poly A and poly U. In particular, the enzymatic activities toward poly I of D51T and D51S were higher than that of RNase RNAP Rh. Among the mutant enzymes, D51N, D51S, and D51T showed more than ca. 30% of the activity of RNase Rh, when RNA, poly I and poly C were used as substrates, respectively. The substitution of Ala, Glu, or Lys at Asp51 is unfavorable for enzymatic activity. Among XpGs (X = A, G, U, or C), D51N, D51S, and D51T showed higher activity toward GpG then CpG. Therefore, Asp51 in RNase Rh plays a critical role in the adenylic acid preference of RNase T2 family enzymes. Our results obtained with a protein engineering technique provide basic insights into the control of the base specificity of RNase Rh.
authors.affiliation: Department of Microbiology, Hoshi College of Pharmacy, Tokyo.
authors.forename: K
authors.initials: K
authors.lastname: Ohgi
authors.forename: M
authors.initials: M
authors.lastname: Takeuchi
authors.forename: M
authors.initials: M
authors.lastname: Iwama
authors.forename: M
authors.initials: M
authors.lastname: Irie
mesh_terms: D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism*; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011131:Polyribonucleotides / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics
chemical_list: D015226:Dinucleoside Phosphates; D011131:Polyribonucleotides; D001224:Aspartic Acid; D012313:RNA; D004722:Endoribonucleases; C075842:ribonuclease Rh