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

• Published in: The Journal of biological chemistry 1994-07, Vol.269 (30), p.19245-19249
• Main Authors: MIZRAHI, V, BROOKSBANK, R. L, NKABINDE, N. C
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Asparagine - genetics; Aspartic Acid - genetics; Base Sequence; Biological and medical sciences; Conserved Sequence; DNA Mutational Analysis; Enzyme Stability - genetics; Enzymes and enzyme inhibitors; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Glutamates - genetics; Glutamic Acid; HIV Reverse Transcriptase; HIV-1 - enzymology; human immunodeficiency virus 1; Hydrolases; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Protein Denaturation; Ribonuclease H - genetics; Ribonuclease H - metabolism; RNA-Directed DNA Polymerase - genetics; RNA-Directed DNA Polymerase - metabolism; Structure-Activity Relationship; Transferases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(17)32159-2

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.