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 nV 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. cali 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 rD134HIRNase H and [D134N]RNase H were shown to have considerable ribonuclease H activities. Determination of the kinetic parameters revealed that replacement of Asp1 34 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 nV helix. These results strongly suggest that Asp134 does not contribute to the maintenance of the molecular architecture but the carboxyl oxygen at its 61 position impacts catalysis.Ribonuclease H (RNase H), which degrades only the RNA strand of an RNA . DNA hybrid in the presence of divalent cations, such as Mg" or Mn", is widely found in various organisms [ 1 -31. Eschericlziu coli RNase HI, which had been designated as RNase H until a second RNase H (RNase HII) was isolated from E. coli 141, is composed of a single polypeptide chain with 155 amino acid residues [S] and has a PI value of 9.0 [6]. The similarities in the primary structures of this enzyme, reverse transcriptase RNase H 17, 81, Thermus thermophilus RNase H [91, and yeast RNase H [lo] strongly suggest that these enzymes are evolutionally related. In fact, the three-dimensional structure of E. coli RNase HI [ll-131 is similar to that of the reverse-transcriptase RNase H from human immunodeficiency virus-1 (HIV-1) [14-161. Therefore, it has been anticipated that detailed analyses of the structure/function relationships of E. Correspondence to S . Kanaya, Protein Engineering ResearchFax: +81 6 872 8210. Abbreviutions. GdnHCI, guanidine hydrochloride; HIV-1, human immunodeficiency virus-1 ; PCR, polymerase chain reaction; t,,*, temperature at which an enzyme loses 50% of its activity; P,, conformational parameter for u-helical residues in proteins ; RNase, ribonuclease; [DI 34XIRNase H, mutant ribonuclease H in which aspartic acid at position 134 is replaced by a specified amino acid.Institute,

Characterization of a folding intermediate from HIV‐1 ribonuclease H

“…The positioning of this loop is clearly dependent on the correct packing of helix E to the rest of the RNase H domain. Similar changes in the homologous E. coli protein affect activity (Oda et al, 1993;Haruki et al, 1994;Goedken et al, 1997). These data suggest that disrupting the docking of helix E onto the rest of the protein would efficiently inactivate the enzyme and prevent viral infectivity.…”

Thermal Stability of Escherichia coli Ribonuclease HI and Its Active Site Mutants in the Presence and Absence of the Mg2+ Ion

“…Site (Kanaya et al, 1990a;Oda et al, 1993;Haruki et al, 1994). 3 Asn mutation suggested that the formation of hydrogen bond networks freezes the catalytic residue or prevents the binding of the Mg 2ϩ ion and thereby inactivates the enzyme (Katayanagi et al, 1993a).…”

Co-crystal of Escherichia coli RNase HI with Mn2+ Ions Reveals Two Divalent Metals Bound in the Active Site

“…This, together with the different stoichiometry observed via crystallography, raises the possibility of alternate binding modes and/or catalytic requirements for these metals. In E. coli RNase HI, conservative mutation of the residues comprising Site 1 (Asp-10, Glu-48, and Asp-70) eliminates activity (12), whereas mutations of the conserved histidine (His-124) (13) and aspartate (Asp-134) of Site 2 (14) have smaller effects on catalysis. Despite structural efforts to identify additional weaker binding sites (5), no metal binding to Site 2 in E. coli RNase HI has been reported.…”