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

Abstract : 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.
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Submitted on : Thursday, November 12, 2009 - 7:44:06 PM
Last modification on : Wednesday, October 14, 2020 - 4:01:56 AM

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Sébastien Graziani, Julie Bernauer, Stéphane Skouloubris, Marc Graille, Cong-Zhao Zhou, et al.. Catalytic mechanism and structure of viral flavin-dependent thymidylate synthase ThyX.. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2006, 281 (33), pp.24048-57. ⟨10.1074/jbc.M600745200⟩. ⟨inria-00431698⟩



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