Catalytic mechanism and structure of viral flavin-dependent thymidylate synthase ThyX.

Archive ouverte

Graziani, Sébastien | Bernauer, Julie | Skouloubris, Stéphane | Graille, Marc | Zhou, Cong-Zhao | Marchand, Christophe | Decottignies, Paulette | van Tilbeurgh, Herman | Myllykallio, Hannu | Liebl, Ursula

Edité par CCSD ; American Society for Biochemistry and Molecular Biology -

International audience. By using biochemical and structural analyses, we have investigated the catalytic mechanism of the recently discovered flavin-dependent thymidylate synthase ThyX from Paramecium bursaria chlorella virus-1 (PBCV-1). Site-directed mutagenesis experiments have identified several residues implicated in either NADPH oxidation or deprotonation activity of PBCV-1 ThyX. Chemical modification by diethyl pyrocarbonate and mass spectroscopic analyses identified a histidine residue (His53) crucial for NADPH oxidation and located in the vicinity of the redox active N-5 atom of the FAD ring system. Moreover, we observed that the conformation of active site key residues of PBCV-1 ThyX differs from earlier reported ThyX structures, suggesting structural changes during catalysis. Steady-state kinetic analyses support a reaction mechanism where ThyX catalysis proceeds via formation of distinct ternary complexes without formation of a methyl enzyme intermediate.

• Description
• Sujet/Public
• Outil
• Outil d'anticipation
• Mémoire et thèse
• Gestion