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International audience; Topoisomerases are essential enzymes that solve topological problems arising from the double-helical structure of DNA. As a consequence, one should have naively expected to find homologous topoisomerases in all cellular organisms, dating back to their last common ancestor. However, as observed for other enzymes working with DNA, this is not the case. Phylogenomics analyses indicate that different sets of topoisomerases were present in the most recent common ancestors of each of the three cellular domains of life (some of them being common to two or three domains), whereas other topoisomerases families or subfamilies were acquired in a particular domain, or even a particular lineage, by horizontal gene transfers. Interestingly, two groups of viruses encode topoisomerases that are only distantly related to their cellular counterparts. To explain these observations, we suggest that topoisomerases originated in an ancestral virosphere, and that various subfamilies were later on transferred independently to different ancient cellular lineages. We also proposed that topoisomerases have played a critical role in the origin of modern genomes and in the emergence of the three cellular domains.

Candida guilliermondii and human DNA topoisomerases I are inhibited by PL (pyridoxal), PLP (pyridoxal 5'-phosphate) and PLP-AMP (pyridoxal 5'-diphospho-5'-adenosine) (PLPLPPLP-AMP). We have recently shown that PLP acted as a competitive inhibitor of C. guilliermondii topoisomerase I, impeding the formation of the cleavable complex from a selective binding to an active site lysine. The targeted lysine in C. guilliermondii topoisomerase I occupies a position equivalent to that of lysine 532 (K(532)) in human topoisomerase I. K(532) acts as a general acid catalyst and is essential for the enzyme activity. This observation has suggested that, in the cell, PLP could down-regulate topoisomerases IB. We have proposed that PLP could be used as a new lead for anticancer drugs trapping the active site lysine (K(532)) and also as a tool to explore the enzyme dynamics required for catalysis. Now we explore the effects of PL, PLP and PLP-AMP on topoisomerases by a molecular modelling approach using the crystal structure of the human topoisomerase I active site and the conformation of K(39)-PLP moiety in Bacillus subtilis alanine racemase as templates. In the modified topoisomerase I several reactive atoms of the K(532)-PLP moiety are at close distance of the catalytic residues R(488), R(590), H(632) and Y(723,) suggesting that PLP develops disturbing interactions with these important residues. These interactions and the corresponding induced fit in the active site conformation are compared with the ones occurring with PL and PLP-AMP. The results could be useful in the search of topoisomerase I inhibitors related to the pyridoxal family.

International audience; 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.