TY  - JOUR
AU  - Czapinska, Honorata
AU  - Siwek, Wojciech
AU  - Szczepanowski, Roman H.
AU  - Bujnicki, Janusz M.
AU  - Bochtler, Matthias
AU  - Skowronek, Krzysztof J.
TI  - Crystal Structure and Directed Evolution of Specificity of NlaIV Restriction Endonuclease
JO  - Journal of molecular biology
VL  - 431
IS  - 11
SN  - 0022-2836
CY  - Amsterdam [u.a.]
PB  - Elsevier
M1  - PUBDB-2019-05186
SP  - 2082 - 2094
PY  - 2019
AB  - 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 kcat/KM 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
LB  - PUB:(DE-HGF)16
C6  - pmid:30995450
UR  - <Go to ISI:>//WOS:000470944400003
DO  - DOI:10.1016/j.jmb.2019.04.010
UR  - https://bib-pubdb1.desy.de/record/429666
ER  -