Identification of the amino acid residues involved in an active site of Escherichia coli ribonuclease H by site-directed mutagenesis.
The amino acid residues essential for the catalytic activity of ribonuclease H (RNase H) from Escherichia coli (E. coli) were identified by site-directed mutagenesis. It has been proposed by computer analysis that E. coli RNase H has homologous amino acid sequence with the RNase H domains of various retroviral reverse transcriptases (RTs) (Johnson, M. S., McClure, M. A., Feng, D. F., Gray, J., and Doolittle, R. F. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7648--7652). Of the eight highly conserved residues examined, Asp10, Glu48, and Asp70 were found to be crucial for RNase H activity. Determination of the kinetic parameters for the mutated enzymes using the chemically synthesized nonanucleotide duplex as a substrate demonstrated that these residues are involved in the catalytic site rather than the substrate-binding site. These residues are fully conserved in the amino acid sequences of not only retroviral RTs but also hepadnaviral, plant viral and retrotransposon RTs. This strongly suggests that they are also involved in the active site of these RTs and RT related enzymes.
Protein Engineering Research Institue, Osaka, Japan.
S
S
Kanaya
A
A
Kohara
Y
Y
Miura
A
A
Sekiguchi
S
S
Iwai
H
H
Inoue
E
E
Ohtsuka
M
M
Ikehara
D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D011487:Protein Conformation; D012194:RNA-Directed DNA Polymerase; D012190:Retroviridae / Q000201:enzymology; D016914:Ribonuclease H; D012689:Sequence Homology, Nucleic Acid; D014169:Transformation, Bacterial
D012194:RNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H