Addressing the challenge of changing the specificity of RNase T1 with rational and evolutionary approaches.
Although ribonuclease T1 (RNase T1) is one of the best-characterized proteins with respect to structure and enzymatic action, numerous attempts at altering the specificity of the enzyme to cleave single-stranded RNA at the 3'-side of adenylic instead of guanylic residues by rational approaches have failed so far. Recently we generated and characterized the RNase T1 variant RV with a 7200-fold increase in adenylyl-3',5'-cytidine (ApC)/guanylyl-3',5'-cytidine (GpC) preference, with the guanine-binding loop changed from 41-KYNNYE-46 (wt) to 41-EFRNWN-46. Now we have introduced the asparagine residue at position 46 of the wild-type enzyme as a single-point mutation in variant E46N and in combination with the Y45W exchange also occurring in RV. Both variants show an improved ApC/GpC preference with a 1450-fold increase for E46N and a 2100-fold increase for Y45W/E46N in comparison to wild-type activity. We also addressed the challenge of altering enzyme specificity with an evolutionary approach. We have randomly introduced point mutations into the RNase T1 wild-type gene and into the gene of the variant RV with different mutation rates. Altogether we have screened about 100,000 individual clones for activity on RNase indicator plates; 533 of these clones were active. A significant change in substrate specificity towards an ApC preference could not be observed for any of these active variants; this demonstrated the magnitude of the challenge to alter the specificity of this evolutionary perfected enzyme.
Struhalla
Marc
M
Institut für Biochemie, Fakultät für Biowissenschaften, Pharmazie und Psychologie, Universität Leipzig, Brüderstrasse 34, 04103 Leipzig, Germany.
Czaja
Rico
R
Hahn
Ulrich
U
D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D032621:DNA Shuffling; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D015202:Protein Engineering; D006163:Ribonuclease T1 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D000596:Amino Acids; D006163:Ribonuclease T1