Investigating the role of conserved residue Asp134 in Escherichia coli ribonuclease HI by site-directed random mutagenesis.


Abstract

The role of the conserved Asp134 residue in Escherichia coli ribonuclease HI, which is located at the center of the alpha V helix and lies close to the active site, was analyzed by means of site-directed random mutagenesis. Mutant rnhA genes encoding proteins with ribonuclease H activities were screened by their ability to suppress the ribonuclease-H-dependent, temperature-sensitive growth phenotype of E. coli strain MIC3001. Based on the DNA sequences, nine mutant proteins were predicted to have ribonuclease H activity in vivo. All of these mutant proteins were purified to homogeneity and examined for enzymic activity and protein stability. Among them, only the mutant proteins [D134H]RNase H and [D134N]RNase H were shown to have considerable ribonuclease H activities. Determination of the kinetic parameters revealed that replacement of Asp134 by amino acid residues other than asparagine and histidine dramatically decreased the enzymic activity without seriously affecting the substrate binding. Determination of the CD spectra indicated that none of the mutations seriously affected secondary and tertiary structure. The protein stability was determined from the thermal denaturation curves. All mutant proteins were more stable than the wild-type protein. Such stabilization effects would be a result of a reduction in the negative charge repulsion between Asp134 and the active-site residues, and/or an enhancement of the stability of the alpha V helix. These results strongly suggest that Asp134 does not contribute to the maintenance of the molecular architecture but the carboxyl oxygen at its delta 1 position impacts catalysis. Study holds ProTherm entries: 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 13351, 13352, 13353, 13354, 13355, 13356, 13357, 13358, 13359, 13360, 13361, 13362, 13363, 13364, 13365, 13366, 13367, 13368, 13369, 13370 Extra Details: Activity determined at 30 degree C

Submission Details

ID: xjXpvFLX3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:15 p.m.

Version: 1

Publication Details
Haruki M;Noguchi E;Nakai C;Liu YY;Oobatake M;Itaya M;Kanaya S,Eur. J. Biochem. (1994) Investigating the role of conserved residue Asp134 in Escherichia coli ribonuclease HI by site-directed random mutagenesis. PMID:8125123
Additional Information

Number of data points 180
Proteins Ribonuclease HI ; Ribonuclease HI ; Ribonuclease HI ; Ribonuclease HI ; Ribonuclease HI
Unique complexes 11
Assays/Quantities/Protocols Experimental Assay: activity temp:50.0 C, pH:3.0, buffers:glycine-HCl: 10 mM ; Experimental Assay: activity_kcat temp:50.0 C, pH:3.0, buffers:glycine-HCl: 10 mM ; Experimental Assay: ddG temp:50.0 C, pH:3.0, buffers:glycine-HCl: 10 mM ; Experimental Assay: activity_km temp:50.0 C, pH:3.0, buffers:glycine-HCl: 10 mM ; Experimental Assay: activity pH:5.5, temp:53.1 C, buffers:Sodium acetate: 20 mM ; Experimental Assay: activity_kcat pH:5.5, temp:53.1 C, buffers:Sodium acetate: 20 mM ; Experimental Assay: ddG pH:5.5, temp:53.1 C, buffers:Sodium acetate: 20 mM ; Experimental Assay: activity_km pH:5.5, temp:53.1 C, buffers:Sodium acetate: 20 mM ; Experimental Assay: activity_kcat pH:3.0, buffers:glycine-HCl: 10 mM ; Experimental Assay: activity pH:3.0, buffers:glycine-HCl: 10 mM ; Experimental Assay: Tm pH:3.0, buffers:glycine-HCl: 10 mM ; Experimental Assay: dHvH pH:3.0, buffers:glycine-HCl: 10 mM ; Experimental Assay: activity_km pH:3.0, buffers:glycine-HCl: 10 mM ; Experimental Assay: activity_kcat pH:5.5, buffers:Sodium acetate: 20 mM ; Experimental Assay: activity pH:5.5, buffers:Sodium acetate: 20 mM ; Experimental Assay: Tm pH:5.5, buffers:Sodium acetate: 20 mM ; Experimental Assay: dHvH pH:5.5, buffers:Sodium acetate: 20 mM ; Experimental Assay: activity_km pH:5.5, buffers:Sodium acetate: 20 mM ; Derived Quantity: dTm pH:3.0, buffers:glycine-HCl: 10 mM ; Derived Quantity: dTm pH:5.5, buffers:Sodium acetate: 20 mM
Libraries Mutations for sequence MLKQVEIFTDGSCLGNPGPGGYGAILRYRGREKTFSAGYTRTTNNRMELMAAIVALEALKEHCEVILSTDSQYVRQGITQWIHNWKKRGWKTADKKPVKNVDLWQRLDAALGQHQIKWEWVKGHAGHPENERCDELARAAAMNPTLEDTGYQVEV
Sequence Assay Result Units