title: The glutamine side chain at position 91 on the β5a-β5b loop of human immunodeficiency virus type 1 reverse transcriptase is required for stabilizing the dNTP binding pocket.
abstract: Earlier, we postulated that Gln91 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) stabilizes the side chain of Tyr183 via hydrogen bonding interaction between O(H) of Tyr183 and CO of Q91 [Harris, D., et al. (1998) Biochemistry 37, 9630-9640]. To test this hypothesis, we generated mutant derivatives of Gln91 and analyzed their biochemical properties. The efficiency of reverse transcription was severely impaired by nonconservative substitution of Gln with Ala, while conservative substitution of Gln with Asn resulted in an approximately 70% loss of activity, a value similar to that observed with the Y183F mutation. The loss of polymerase activity from both Q91A and Q91N was significantly improved by a Met to Val substitution at position 184. Curiously, the Q91N mutant exhibited stringency in discriminating between correct and incorrect nucleotides, suggesting its possible interaction with residues influencing the flexibility of the dNTP binding pocket. In contrast, both double mutants, Q91A/M184V and Q91N/M184V, are found to be as error prone as the wild-type enzyme. We propose a model that suggests that subtle structural changes in the region due to mutation at position 91 may influence the stability of the side chain of Tyr183 in the catalytic YMDD motif of the enzyme, thus altering the active site geometry that may interfere in substrate recognition.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103, USA.
authors.forename: Nootan
authors.initials: N
authors.lastname: Pandey
authors.forename: Chaturbhuj A
authors.initials: CA
authors.lastname: Mishra
authors.forename: Dinesh
authors.initials: D
authors.lastname: Manvar
authors.forename: Alok K
authors.initials: AK
authors.lastname: Upadhyay
authors.forename: Tanaji T
authors.initials: TT
authors.lastname: Talele
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Comollo
authors.forename: Neerja
authors.initials: N
authors.lastname: Kaushik-Basu
authors.forename: Virendra N
authors.initials: VN
authors.lastname: Pandey
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray / Q000379:methods; D003854:Deoxyribonucleotides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D055550:Protein Stability; D017433:Protein Structure, Secondary; D013379:Substrate Specificity / Q000235:genetics; D014443:Tyrosine / Q000378:metabolism
chemical_list: D003854:Deoxyribonucleotides; D005973:Glutamine; D014443:Tyrosine; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase