Stabilization of Moloney murine leukemia virus reverse transcriptase by site-directed mutagenesis of surface residue Val433

Abstract

After thermal incubation at 48 °C for 10 min, single variants of Moloney murine leukemia virus reverse transcriptase, V433R and V433K in which a surface hydrophobic residue, Val433, was mutated, retained 55% of initial reverse transcription activity, while the wild-type enzyme retained 17%. After thermal incubation at 50 °C for 10 min, multiple variants D108R/E286R/V433R and D108R/E286R/V433R/D524A, in which Val433→Arg was combined with stabilizing mutations we identified previously, Asp108→Arg and Glu286→Arg, and RNase H activity-eliminating mutation Asp524→Ala, retained 70% of initial activity, exhibiting higher stability than V433R or V433K.

Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system

“…[9][10][11] We improved the thermostability of MMLV RT by sitedirected mutagenesis and generated MM4 (E286R/ E302K/L435R/D524A) in which negatively charged (Glu286, Glu302) or hydrophobic (Leu435) residues thought to interact with a template-primer were replaced with positively charged ones and the catalytic residue for RNase H activity (Asp524) was replaced with Ala. 12) We also generated V433R in which Val433 at the molecular surface was replaced with Arg. 13) Recently, we designed 29 mutations, focusing on the number of surface charge and stabilization of hydrophobic core, and finally generated a sextuple thermostable variant MM3.14 (A32V/ L72R/E286R/E302K/W388R/L435R). 14) Others improved thermostability of MMLV RT by random mutagenesis, in which error-prone PCR was used for the construction of MMLV RT gene library, and filter assay 15) or emulsion PCR 16) was used for the screening of MMLV RT with higher thermostability.…”

Alteration of enzymes and their application to nucleic acid amplification (Review)

“…The thermostability of MMLV RT was improved by introducing the triple mutation E286R/E302K/L435R or E286R/E302K/L435R/d524A in which the negatively charged (Glu286 and Glu302) and hydrophobic (Leu435) residues that were thought to interact with a template-primer were replaced with positively charged residues, and the catalytic residue responsible for RNase H activity Asp524 was replaced with Ala (22). The thermostability of MMLV RT was also improved by the mutation of Val433 present on the molecular surface to Arg (23). Finally, a highly thermostable MMLV variant A32V/L72R/E286R/E302K/W388R/L435R was generated by combining the triple mutation E286R/E302K/L435R with the following mutations: The mutation of the internal residue, Ala32 to Val in order to stabilize the hydrophobic core, the mutation of the hydrophobic surface residue, Leu72 to Arg, and the mutation of Trp388 which is close to the negatively charged residues to Arg in order to introduce a salt bridge (24).…”

Amino acid substitutions away from the RNase H catalytic site increase the thermal stability of Moloney murine leukemia virus reverse transcriptase through RNase H inactivation

“…Improving the efficiency of the RT DNA polymerase activity at high temperatures has been an important area of research in biotechnology. Available RTs efficient at high temperatures have been obtained by inactivating their RNase H activity [4][5][6][7][8], or by improving template-primer binding affinity [9][10][11]. Random mutagenesis [12,13] and rational design [9][10][11] have been used to generate those RT variants.…”

“…Available RTs efficient at high temperatures have been obtained by inactivating their RNase H activity [4][5][6][7][8], or by improving template-primer binding affinity [9][10][11]. Random mutagenesis [12,13] and rational design [9][10][11] have been used to generate those RT variants. Thermostable RTs from MMLV [4][5][6], avian myeloblastosis virus (AMV) [5,7], and human immunodeficiency virus type 1 (HIV-1) [8] have been generated by deleting the RNase H domain [5] or by site-directed mutagenesis of the catalytic residues of the RNase H activity [6][7][8].…”

“…We have previously identified five stabilizing mutations (E286R, E302K, L435R, V433K, and V433R) [9,10] by introducing basic residues in the nucleic acid binding cleft of the RT. In those studies, we hypothesized that the introduction of positive charges increases the thermostability of MMLV RT by improving its ability to bind the T/P that is negatively charged.…”