The restriction endonuclease
EcoRV has been characterized instructural and functional terms in great detail. Based on thisdetailed information we employed a structure-guided approachto engineer variants of
EcoRV that should be able to discriminatebetween differently flanked
EcoRV recognition sites. In crystalstructures of
EcoRV complexed with d(CGGGATATCCC)
2 and d(AAAGATATCTT)
2,Lys104 and Ala181 closely approach the two base pairs flankingthe GATATC recognition site and thus were proposed to be a reasonablestarting point for the rational extension of site specificityin
EcoRV [Horton,N.C. and Perona,J.J. (1998)
J. Biol. Chem.,273, 2172121729]. To test this proposal, several single(K104R, A181E, A181K) and double mutants of
EcoRV (K104R/A181E,K104R/A181K) were generated. A detailed characterization ofall variants examined shows that only the substitution of Ala181by Glu leads to a considerably altered selectivity with botholigodeoxynucleotide and macromolecular DNA substrates, butnot the predicted one, as these variants prefer cleavage ofa TA flanked site over all other sites, under all conditionstested. The substitution of Lys104 by Arg, in contrast, whichappeared to be very promising on the basis of the crystallographicanalysis, does not lead to variants which differ very much fromthe
EcoRV wild-type enzyme with respect to the flanking sequencepreferences. The K104R/A181E and K104R/A181K double mutantsshow nearly the same preferences as the A181E and A181K singlemutants. We conclude that even for the very well characterizedrestriction enzyme
EcoRV, properties that determine specificityand selectivity are difficult to model on the basis of the availablestructural information.
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