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Nucleic Acids Research 2004 32(18):5649-5657; doi:10.1093/nar/gkh897
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Published online 19 October 2004

Nucleic Acids Research, Vol. 32 No. 18 © Oxford University Press 2004; all rights reserved

Pyridoxal 5'-phosphate inactivates DNA topoisomerase IB by modifying the lysine general acid

Jacqueline J. Vermeersch1, Serge Christmann-Franck2,3, Leon V. Karabashyan4, Serge Fermandjian2, Gilles Mirambeau5 and P. Arsène Der Garabedian1,*

1 Biochimie des Signaux Régulateurs Cellulaires et Moléculaires, Université Pierre et Marie Curie, CNRS FRE 2621, 96 Boulevard Raspail, 75006 Paris, France, 2 Département de Biologie et Pharmacologie Structurales, ENS Cachan, CNRS UMR 8113, 61 Avenue du Président Wilson, 94235 Cachan cedex, France, 3 Accelrys, 20 Rue Jean Rostand, 91898 Orsay cedex, France, 4 Institute of Molecular Biology, National Academy Science of Armenia, 7 Hasratyan Street, Yerevan, Armenia and 5 CNRS UMR 8126, Institut Gustave Roussy, 94805 Villejuif cedex, France

* To whom correspondence should be addressed. Tel: +331 53 63 40 76; Fax: +331 53 63 40 77; Email: dergara{at}ccr.jussieu.fr

Received May 17, 2004; Revised July 16, 2004; Accepted September 30, 2004

The present results demonstrate that pyridoxal, pyridoxal 5'-phosphate (PLP) and pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) inhibit Candida guilliermondii and human DNA topoisomerases I in forming an aldimine with the {epsilon}-amino group of an active site lysine. PLP acts as a competitive inhibitor of C.guilliermondii topoisomerase I (Ki = 40 µM) that blocks the cleavable complex formation. Chemical reduction of PLP-treated enzyme reveals incorporation of 1 mol of PLP per mol of protein. The limited trypsic proteolysis releases a 17 residue peptide bearing a lysine-bound PLP (KPPNTVIFDFLGK*DSIR). Targeted lysine (K*) in C.guilliermondii topoisomerase I corresponds to that found in topoisomerase I of Homo sapiens (K532), Candida albicans (K468), Saccharomyces cerevisiae (K458) and Schizosaccharomyces pombe (K505). In the human enzyme, K532, belonging to the active site acts as a general acid catalyst and is therefore essential for activity. The spatial orientation of K532–PLP within the active site was approached by molecular modeling using available crystallographic data. The PLP moiety was found at close proximity of several active residues. PLP could be involved in the cellular control of topoisomerases IB. It constitutes an efficient tool to explore topoisomerase IB dynamics during catalysis and is also a lead for new drugs that trap the lysine general acid.


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