title: Increase of specificity of RNase from Bacillus amyloliquefaciens (barnase) by substitution of Glu for Ser57 using site-directed mutagenesis.
abstract: Bacterial ribonucleases from Bacillus amyloliquefaciens and Bacillus intermedius show the specificity towards the nature of a nucleoside at the O3' end of the phosphodiester 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 T1.
authors.affiliation: Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow.
authors.forename: G I
authors.initials: GI
authors.lastname: Yakovlev
authors.forename: G P
authors.initials: GP
authors.lastname: Moiseyev
authors.forename: N K
authors.initials: NK
authors.lastname: Struminskaya
authors.forename: E R
authors.initials: ER
authors.lastname: Romakhina
authors.forename: I B
authors.initials: IB
authors.lastname: Leshchinskaya
authors.forename: R W
authors.initials: RW
authors.lastname: Hartley
mesh_terms: D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins; D001483:Base Sequence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease