Open AccessIncrease of specificity of RNase from Bacillus amyloliquefaciens (barnase) by substitution of Glu for Ser57 using site‐directed mutagenesis
Author(s) -
YAKOVLEV Gennady I.,
MOISEYEV Gennady P.,
STRUMINSKAYA Nina K.,
ROMAKHINA Elena R.,
LESHCHINSKAYA Inna B.,
HARTLEY Robert W.
Publication year - 1993
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1993.tb18019.x
Subject(s) - barnase , bacillus amyloliquefaciens , rnase p , mutagenesis , site directed mutagenesis , chemistry , substitution (logic) , saturated mutagenesis , biochemistry , mutation , ribonuclease , computer science , gene , rna , mutant , fermentation , programming language
Bacterial ribonucleases from Bacillus amyloliquefaciens and Bacillus intermedius show the specificity towards the nature of a nucleoside at the O3′ end of the phoshodiester bond to be split in the preference order G> A≫ U>C in the cleavage reactions of polynucleotides. It follows from the X‐ray data that the substrate guanosine base is bound at the active site of these RNases in the same manner as for high‐specificity guanylic RNases. We supposed that the difference in specificity for the two types of RNases is due to the additional hydrogen bond between the protein and a purine base in the case of bacterial guanyl‐preferring RNases in contrast to the high‐specificity guanylic RNases. To examine this hypothesis we prepared the Ser57→Glu mutant of B. amyloliquefaciens , in which this hydrogen bond is eliminated. Kinetic studies demonstrate that the specificity of this mutant towards guanylic substrates is 35‐times greater than that of the wild‐type RNases from B. amyloliquefaciens and close to that of RNases T 1 .