ThyX (Paramecium bursaria chlorella virus-1)

(JPEG)
A, ribbon representation of the PBCV-1 ThyX monomer. B, ribbon representation of the ThyX homotetramer; each monomer is colored differently. The four bound FAD molecules are shown as sticks. The monomer highlighted in green is related to the representation in A by a 90° rotation along the x axis. C, stereo view representation of the comparison of the ThyX FAD binding mode. PBCV-1 and MtbThyX are colored yellow and green, respectively. For clarity, only PBCV-1 ThyX numbering is indicated. As TmThyX FAD binding mode is closely similar to that of MtbThyX, it has been omitted. Phosphorous atoms from FAD are colored black. For clarity, the labels corresponding to residues from monomer B are underlined. D, stereo view representation of the superposition of the active sites from PBCV-1 (yellow), MtbThyX (green), and TmThyX (gray). For clarity, only PBCV-1 ThyX numbering is indicated. Phosphorous atoms from FAD are colored black. E, ribbon representation of the hydrogen bonding pattern responsible for the binding of the FAD isoalloxazine moiety. Residues from monomers A, A’, and B are colored gray, yellow, and green, respectively.

Function Thymidylate synthase
Fold alpha/beta domain
Resolution 2.3
Remarks Complex with FAD
Biological unit Tetramer
PDB code 2CFA
Reference Graziani, S., Bernauer, J., Skouloubris, S., Graille, M., Zhou, C.-Z., Marchand, C., Decottignies, P., Van Tilbeurgh, H., Myllykallio, H., Liebl, U. Catalytic Mechanism and Structure of Viral Flavin-Dependent Thymidylate Synthase Thyx. J.Biol.Chem. v281 pp.24048 , 2006 Full text

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