Home > Publications database > Crystal Structure and Directed Evolution of Specificity of NlaIV Restriction Endonuclease > print

001     429666
005     20250729163231.0
024 7 _ |a 10.1016/j.jmb.2019.04.010
|2 doi
024 7 _ |a 0022-2836
|2 ISSN
024 7 _ |a 1089-8638
|2 ISSN
024 7 _ |a 10.3204/PUBDB-2019-05186
|2 datacite_doi
024 7 _ |a pmid:30995450
|2 pmid
024 7 _ |a WOS:000470944400003
|2 WOS
024 7 _ |a altmetric:126186206
|2 altmetric
024 7 _ |a openalex:W2940309740
|2 openalex
037 _ _ |a PUBDB-2019-05186
041 _ _ |a English
082 _ _ |a 610
100 1 _ |a Czapinska, Honorata
|0 P:(DE-H253)PIP1011581
|b 0
245 _ _ |a Crystal Structure and Directed Evolution of Specificity of NlaIV Restriction Endonuclease
260 _ _ |a Amsterdam [u.a.]
|c 2019
|b Elsevier
336 7 _ |a article
|2 DRIVER
336 7 _ |a Output Types/Journal article
|2 DataCite
336 7 _ |a Journal Article
|b journal
|m journal
|0 PUB:(DE-HGF)16
|s 1576664913_29636
|2 PUB:(DE-HGF)
336 7 _ |a ARTICLE
|2 BibTeX
336 7 _ |a JOURNAL_ARTICLE
|2 ORCID
336 7 _ |a Journal Article
|0 0
|2 EndNote
520 _ _ |a 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
536 _ _ |a 899 - ohne Topic (POF3-899)
|0 G:(DE-HGF)POF3-899
|c POF3-899
|f POF III
|x 0
588 _ _ |a Dataset connected to CrossRef
693 _ _ |a DORIS III
|f DORIS Beamline BW6
|1 EXP:(DE-H253)DORISIII-20150101
|0 EXP:(DE-H253)D-BW6-20150101
|6 EXP:(DE-H253)D-BW6-20150101
|x 0
700 1 _ |a Siwek, Wojciech
|0 P:(DE-H253)PIP1012258
|b 1
700 1 _ |a Szczepanowski, Roman H.
|b 2
700 1 _ |a Bujnicki, Janusz M.
|0 P:(DE-H253)PIP1028870
|b 3
700 1 _ |a Bochtler, Matthias
|0 P:(DE-H253)PIP1011590
|b 4
700 1 _ |a Skowronek, Krzysztof J.
|b 5
773 _ _ |a 10.1016/j.jmb.2019.04.010
|g Vol. 431, no. 11, p. 2082 - 2094
|0 PERI:(DE-600)1355192-9
|n 11
|p 2082 - 2094
|t Journal of molecular biology
|v 431
|y 2019
|x 0022-2836
856 4 _ |y OpenAccess
|u https://bib-pubdb1.desy.de/record/429666/files/2019_czapinska.pdf
856 4 _ |y OpenAccess
|x icon
|u https://bib-pubdb1.desy.de/record/429666/files/2019_czapinska.gif?subformat=icon
856 4 _ |y OpenAccess
|x icon-1440
|u https://bib-pubdb1.desy.de/record/429666/files/2019_czapinska.jpg?subformat=icon-1440
856 4 _ |y OpenAccess
|x icon-180
|u https://bib-pubdb1.desy.de/record/429666/files/2019_czapinska.jpg?subformat=icon-180
856 4 _ |y OpenAccess
|x icon-640
|u https://bib-pubdb1.desy.de/record/429666/files/2019_czapinska.jpg?subformat=icon-640
856 4 _ |y OpenAccess
|x pdfa
|u https://bib-pubdb1.desy.de/record/429666/files/2019_czapinska.pdf?subformat=pdfa
909 C O |o oai:bib-pubdb1.desy.de:429666
|p openaire
|p open_access
|p VDB
|p driver
|p dnbdelivery
910 1 _ |a European Molecular Biology Laboratory
|0 I:(DE-588b)235011-7
|k EMBL
|b 0
|6 P:(DE-H253)PIP1011581
910 1 _ |a External Institute
|0 I:(DE-HGF)0
|k Extern
|b 0
|6 P:(DE-H253)PIP1011581
910 1 _ |a External Institute
|0 I:(DE-HGF)0
|k Extern
|b 1
|6 P:(DE-H253)PIP1012258
910 1 _ |a External Institute
|0 I:(DE-HGF)0
|k Extern
|b 3
|6 P:(DE-H253)PIP1028870
910 1 _ |a External Institute
|0 I:(DE-HGF)0
|k Extern
|b 4
|6 P:(DE-H253)PIP1011590
913 1 _ |a DE-HGF
|b Programmungebundene Forschung
|l ohne Programm
|1 G:(DE-HGF)POF3-890
|0 G:(DE-HGF)POF3-899
|2 G:(DE-HGF)POF3-800
|v ohne Topic
|x 0
|4 G:(DE-HGF)POF
|3 G:(DE-HGF)POF3
914 1 _ |y 2019
915 _ _ |a DBCoverage
|0 StatID:(DE-HGF)0200
|2 StatID
|b SCOPUS
915 _ _ |a DBCoverage
|0 StatID:(DE-HGF)1030
|2 StatID
|b Current Contents - Life Sciences
915 _ _ |a DBCoverage
|0 StatID:(DE-HGF)0600
|2 StatID
|b Ebsco Academic Search
915 _ _ |a Creative Commons Attribution-NonCommercial-NoDerivs CC BY-NC-ND 4.0
|0 LIC:(DE-HGF)CCBYNCND4
|2 HGFVOC
915 _ _ |a JCR
|0 StatID:(DE-HGF)0100
|2 StatID
|b J MOL BIOL : 2017
915 _ _ |a DBCoverage
|0 StatID:(DE-HGF)0150
|2 StatID
|b Web of Science Core Collection
915 _ _ |a WoS
|0 StatID:(DE-HGF)0110
|2 StatID
|b Science Citation Index
915 _ _ |a WoS
|0 StatID:(DE-HGF)0111
|2 StatID
|b Science Citation Index Expanded
915 _ _ |a IF < 5
|0 StatID:(DE-HGF)9900
|2 StatID
915 _ _ |a OpenAccess
|0 StatID:(DE-HGF)0510
|2 StatID
915 _ _ |a Peer Review
|0 StatID:(DE-HGF)0030
|2 StatID
|b ASC
915 _ _ |a DBCoverage
|0 StatID:(DE-HGF)0310
|2 StatID
|b NCBI Molecular Biology Database
915 _ _ |a DBCoverage
|0 StatID:(DE-HGF)1050
|2 StatID
|b BIOSIS Previews
915 _ _ |a DBCoverage
|0 StatID:(DE-HGF)0300
|2 StatID
|b Medline
915 _ _ |a Nationallizenz
|0 StatID:(DE-HGF)0420
|2 StatID
915 _ _ |a DBCoverage
|0 StatID:(DE-HGF)0199
|2 StatID
|b Clarivate Analytics Master Journal List
920 1 _ |0 I:(DE-H253)HAS-User-20120731
|k DOOR ; HAS-User
|l DOOR-User
|x 0
980 _ _ |a journal
980 _ _ |a VDB
980 _ _ |a UNRESTRICTED
980 _ _ |a I:(DE-H253)HAS-User-20120731
980 1 _ |a FullTexts

LibraryCollectionCLSMajorCLSMinorLanguageAuthor
Marc 21