Document Detail

Controlling the enzymatic activity of a restriction enzyme by light.
MedLine Citation:
PMID:  20080559     Owner:  NLM     Status:  MEDLINE    
For many applications it would be desirable to be able to control the activity of proteins by using an external signal. In the present study, we have explored the possibility of modulating the activity of a restriction enzyme with light. By cross-linking two suitably located cysteine residues with a bifunctional azobenzene derivative, which can adopt a cis- or trans-configuration when illuminated by UV or blue light, respectively, enzymatic activity can be controlled in a reversible manner. To determine which residues when cross-linked show the largest "photoswitch effect," i.e., difference in activity when illuminated with UV vs. blue light, > 30 variants of a single-chain version of the restriction endonuclease PvuII were produced, modified with azobenzene, and tested for DNA cleavage activity. In general, introducing single cross-links in the enzyme leads to only small effects, whereas with multiple cross-links and additional mutations larger effects are observed. Some of the modified variants, which carry the cross-links close to the catalytic center, can be modulated in their DNA cleavage activity by a factor of up to 16 by illumination with UV (azobenzene in cis) and blue light (azobenzene in trans), respectively. The change in activity is achieved in seconds, is fully reversible, and, in the case analyzed, is due to a change in V(max) rather than K(m).
Benno Schierling; Ann-Josée Noël; Wolfgang Wende; Le Thi Hien; Eugeny Volkov; Elena Kubareva; Tatiana Oretskaya; Michael Kokkinidis; Andreas Römpp; Bernhard Spengler; Alfred Pingoud
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2009-12-18
Journal Detail:
Title:  Proceedings of the National Academy of Sciences of the United States of America     Volume:  107     ISSN:  1091-6490     ISO Abbreviation:  Proc. Natl. Acad. Sci. U.S.A.     Publication Date:  2010 Jan 
Date Detail:
Created Date:  2010-02-05     Completed Date:  2010-03-12     Revised Date:  2013-05-31    
Medline Journal Info:
Nlm Unique ID:  7505876     Medline TA:  Proc Natl Acad Sci U S A     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1361-6     Citation Subset:  IM    
Institute of Biochemistry, Justus-Liebig University, Giessen, Germany.
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MeSH Terms
Alternative Splicing
Azo Compounds / chemistry
Binding Sites
Cross-Linking Reagents / chemistry
Crystallography, X-Ray
DNA Restriction Enzymes / chemistry*,  genetics,  metabolism*
Enzyme Activation / radiation effects
Models, Molecular
Protein Binding
Protein Interaction Domains and Motifs
Protein Structure, Tertiary
Ultraviolet Rays*
Reg. No./Substance:
0/Azo Compounds; 0/Cross-Linking Reagents; EC 3.1.21.-/DNA Restriction Enzymes; F0U1H6UG5C/azobenzene
Comment In:
Proc Natl Acad Sci U S A. 2010 Jan 26;107(4):1259-60   [PMID:  20133886 ]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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