Abstract：
:Attempts to modify the guanine specificity of ribonuclease T1 (RNase T1) by rationally designed amino acid substitutions failed so far. Therefore, we applied a semirational approach by randomizing the guanine binding site. A combinatorial library of approximately 1.6 million RNase T1 variants containing permutations of 6 amino acid positions within the recognition loop was screened on RNase indicator plates. The specificity profiles of 180 individual clones showing RNase activity revealed that variant K41S/N43W/N44H/Y45A/E46D (RNaseT1-8/3) exhibits an altered preference toward purine nucleotides. The ApC/GpC preference in the cleavage reaction of this variant was increased 4000-fold compared to wild-type. Synthesis experiments of dinucleoside monophosphates from cytidine and the corresponding 2'3'-cyclic diesters using the reverse reaction of the transesterification step showed a 7-fold higher ApC synthesis rate of RNase 8/3 than wild-type, whereas the GpC synthesis rates for both enzymes were comparable. This study shows that site-directed random mutagenesis is a powerful additional tool in protein design in order to achieve new enzymatic specificities.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Hubner B,Haensler M,Hahn Udoi
10.1021/bi9817515subject
Has Abstractpub_date
1999-01-26 00:00:00pages
1371-6issue
4eissn
0006-2960issn
1520-4995pii
bi9817515journal_volume
38pub_type
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