Catalytic mechanism and structure of viral flavin-dependent thymidylate synthase ThyX.
J Biol Chem
; 281(33): 24048-57, 2006 Aug 18.
Article
em En
| MEDLINE
| ID: mdl-16707489
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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Base de dados:
MEDLINE
Assunto principal:
Timidilato Sintase
/
Chlorella
/
Flavina-Adenina Dinucleotídeo
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
2006
Tipo de documento:
Article
País de afiliação:
França