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@ARTICLE{Czapinska:429666,
      author       = {Czapinska, Honorata and Siwek, Wojciech and Szczepanowski,
                      Roman H. and Bujnicki, Janusz M. and Bochtler, Matthias and
                      Skowronek, Krzysztof J.},
      title        = {{C}rystal {S}tructure and {D}irected {E}volution of
                      {S}pecificity of {N}la{IV} {R}estriction {E}ndonuclease},
      journal      = {Journal of molecular biology},
      volume       = {431},
      number       = {11},
      issn         = {0022-2836},
      address      = {Amsterdam [u.a.]},
      publisher    = {Elsevier},
      reportid     = {PUBDB-2019-05186},
      pages        = {2082 - 2094},
      year         = {2019},
      abstract     = {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},
      cin          = {DOOR ; HAS-User},
      ddc          = {610},
      cid          = {I:(DE-H253)HAS-User-20120731},
      pnm          = {899 - ohne Topic (POF3-899)},
      pid          = {G:(DE-HGF)POF3-899},
      experiment   = {EXP:(DE-H253)D-BW6-20150101},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:30995450},
      UT           = {WOS:000470944400003},
      doi          = {10.1016/j.jmb.2019.04.010},
      url          = {https://bib-pubdb1.desy.de/record/429666},
}