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Solution structure and DNA binding of the catalytic domain of the large serine resolvase TnpX
Author(s) -
Headey Stephen J.,
Sivakumaran Andrew,
Adams Vicki,
Lyras Dena,
Rood Julian I.,
Scanlon Martin J.,
Wilce Matthew C. J.
Publication year - 2015
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/jmr.2446
Subject(s) - recombinase , site specific recombination , active site , tn3 transposon , chemistry , dna , serine , docking (animal) , plasmid , stereochemistry , binding site , biochemistry , transposable element , recombination , gene , enzyme , mutant , medicine , nursing
The transfer of antibiotic resistance between bacteria is mediated by mobile genetic elements such as plasmids and transposons. TnpX is a member of the large serine recombinase subgroup of site‐specific recombinases and is responsible for the excision and insertion of mobile genetic elements that encode chloramphenicol resistance in the pathogens Clostridium perfringens and Clostridium difficile . TnpX consists of three structural domains: domain I contains the catalytic site, whereas domains II and III contain DNA‐binding motifs. We have solved the solution structure of residues 1–120 of the catalytic domain I of TnpX. The TnpX catalytic domain shares the same overall fold as other serine recombinases; however, differences are evident in the identity of the proposed hydrogen donor and in the size, amino acid composition, conformation, and dynamics of the TnpX active site loops. To obtain the interaction surface of TnpX 1–120 , we titrated a DNA oligonucleotide containing the circular intermediate joint attCI recombination site into 15 N‐labeled TnpX 1–120 and observed progressive nuclear magnetic resonance chemical shift perturbations using 15 N HSQC spectra. Perturbations were largely confined to a region surrounding the catalytic serine and encompassed residues of the active site loops. Utilizing the perturbation map and the data‐driven docking program, HADDOCK, we have generated a model of the DNA interaction complex for the TnpX catalytic domain. Copyright © 2015 John Wiley & Sons, Ltd.