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Catalytic Mechanism and Structure of Viral Flavin-dependent Thymidylate Synthase ThyX.
- Source :
-
Journal of Biological Chemistry . 8/18/2006, Vol. 281 Issue 33, p24048-24057. 10p. 5 Diagrams, 1 Chart, 1 Graph. - Publication Year :
- 2006
-
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, [ABSTRACT FROM AUTHOR]
- Subjects :
- *FLAVINS
*PARAMECIUM
*CATALYSIS
*ENZYMES
*COENZYMES
*PIGMENTS
Subjects
Details
- Language :
- English
- ISSN :
- 00219258
- Volume :
- 281
- Issue :
- 33
- Database :
- Academic Search Index
- Journal :
- Journal of Biological Chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 22326785
- Full Text :
- https://doi.org/10.1074/jbc.M600745200