Mutagenesis of the conserved aspartic acid 443, glutamic acid 478, asparagine 494, and aspartic acid 498 residues in the ribonuclease H domain of p66/p51 human immunodeficiency virus type I reverse transcriptase. Expression and biochemical analysis.
The effects of point mutations of the conserved Asp443, Glu478, Asn494, and Asp498 residues in the RNase H domain of human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) have been analyzed. The mutants fell into two classes: (i) functional RT, but not detectable ribonuclease H activity, and (ii) uncharacterizable phenotype due to protein instability in the context of the RT/protease Escherichia coli co-expression system (Mizrahi, V., Lazarus, G. M., Miles, L. M., Meyers, C. A., and Debouck, C. (1989) Arch. Biochem. Biophys. 273, 347-358). The only mutation in the former class was D443A, whereas those in the latter included D443E, E478D, E478Q, D498E, D443A/D498N, D443E/D498N, D443Q/D498N, N494A, N494D, and N494Q. The results were interpreted in terms of the x-ray crystal structure of the HIV-1 RNase H domain (Davies, J. F., II, Hostomaska, Z., Hostomsky, Z., Jordan, S. R., and Matthews, D. A. (1991) Science 252, 88-95) and a general acid-general base hydrolysis mechanism (Katayanagi, K., Okumura, M., and Morikawa, K. (1993) Proteins Struct. Funct. Genet. 17, 337-346). The data suggested that structural perturbations within the RNase H domain interfered with maturation of the pol precursor by HIV-1 protease. Analysis of selected D443/D498 double mutants suggested that the destabilization caused by the D498N mutation could be suppressed by the formation of a new hydrogen bond between Asn498 and Asn443.
Mizrahi
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Molecular Biology Unit, South African Institute for Medical Research, Johannesburg.
Brooksbank
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RL
Nkabinde
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NC
D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001483:Base Sequence; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005971:Glutamates / Q000235:genetics; D018698:Glutamic Acid; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D005971:Glutamates; D001224:Aspartic Acid; D018698:Glutamic Acid; D001216:Asparagine; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H