부산대학교 도서관

Specificity engineering is challenging and particularly difficult for enzymes that have the catalytic machinery and specificity determinants in close proximity. Restriction endonucleases have been used as a paradigm for protein engineering, but successful cases are rare. Here, we present the results of a directed evolution approach to the engineering of a dimeric, blunt end cutting restriction enzyme NlaIV (GGN/NCC). Based on the remote similarity to EcoRV endonuclease, regions for random mutagenesis and in vitro evolution were chosen. The obtained variants cleaved target sites with an up to 100-fold k cat / K M preference for AT or TA (GGW/WCC) over GC or CG (GGS/SCC) in the central dinucleotide step, compared to the only ~ 17-fold preference of the wild-type enzyme. To understand the basis of the increased specificity, we determined the crystal structure of NlaIV. Despite the presence of DNA in the crystallization mix, the enzyme crystallized in the free form. We therefore constructed a computational model of the NlaIV–DNA complex. According to the model, the mutagenesis of the regions that were in the proximity of DNA did not lead to the desired specificity change, which was instead conveyed in an indirect manner by substitutions in the more distant regions. Unlabelled Image • Specificity engineering of restriction endonucleases is extremely challenging. • Directed evolution was used to re-engineer restriction enzyme specificity. • NlaIV variants with up to 100-fold preference for GGW/WCC over GGS/SCC were obtained. • The crystal structure of NlaIV restriction endonuclease in the apo form was determined. • Substitutions altered the specificity via k cat effects and indirect contacts to DNA. [ABSTRACT FROM AUTHOR]