Catalytic mechanism and structure of viral flavin-dependent thymidylate synthase ThyX.
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.
Graziani
Sébastien
S
CNRS, UMR 7645, Laboratory of Optics and Biosciences, Ecole Polytechnique, 91128 Palaiseau, France.
Bernauer
Julie
J
Skouloubris
Stéphane
S
Graille
Marc
M
Zhou
Cong-Zhao
CZ
Marchand
Christophe
C
Decottignies
Paulette
P
van Tilbeurgh
Herman
H
Myllykallio
Hannu
H
Liebl
Ursula
U
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000378:metabolism; D002384:Catalysis; D002708:Chlorella / Q000821:virology*; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism / Q000502:physiology*; D018698:Glutamic Acid / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D017862:Phycodnaviridae / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*
D004791:Enzyme Inhibitors; D005182:Flavin-Adenine Dinucleotide; D018698:Glutamic Acid; D006639:Histidine; D001120:Arginine; D013940:Thymidylate Synthase; D004047:Diethyl Pyrocarbonate