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.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Czapinska H,Siwek W,Szczepanowski RH,Bujnicki JM,Bochtler M,Skowronek KJdoi
10.1016/j.jmb.2019.04.010subject
Has Abstractpub_date
2019-05-17 00:00:00pages
2082-2094issue
11eissn
0022-2836issn
1089-8638pii
S0022-2836(19)30204-9journal_volume
431pub_type
杂志文章abstract：:Protein catalyzed DNA rearrangements typically require assembly of complex nucleoprotein structures. In transposition and integration reactions, these structures, termed synaptic complexes, are mandatory for catalysis. We characterize the Tn5 pre-cleavage synaptic complex, the simplest transposition complex described ...
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