Pyridoxal 5'-phosphate inactivates DNA topoisomerase IB by modifying the lysine general acid.
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 (K(i) = 40 microM) 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.
Vermeersch
Jacqueline J
JJ
Biochimie des Signaux Régulateurs Cellulaires et Moléculaires, Université Pierre et Marie Curie, CNRS FRE 2621, 96 Boulevard Raspail, 75006 Paris, France.
Christmann-Franck
Serge
S
Karabashyan
Leon V
LV
Fermandjian
Serge
S
Mirambeau
Gilles
G
Der Garabedian
P Arsène
PA
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D001665:Binding Sites; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000187:drug effects* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000187:drug effects*; D008958:Models, Molecular; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D004264:DNA Topoisomerases, Type I; C048236:pyridoxal 5'-diphospho-5'-adenosine; D008239:Lysine