Functional analysis of FAD-dependent thymidylate synthase ThyX from Paramecium bursaria Chlorella virus-1.
Sequence analysis of the 330-kb double-stranded DNA genome of Paramecium bursaria chlorella virus-1 revealed an open reading frame A674R that encodes a protein with up to 53% amino acid identity to a recently discovered new class of thymidylate synthases, called ThyX. Unlike the traditional thymidylate synthase, ThyA, that uses methylenetetrahydrofolate (CH(2)H(4)folate) as both a source of the methylene group and the reductant, CH(2)H(4)folate only supplies the methylene group in ThyX-catalyzed reactions. Furthermore, ThyX only catalyzes thymidylate (dTMP) formation in the presence of reduced pyridine nucleotides and oxidized FAD. The distribution and transcription patterns of the a674r gene in Chlorella viruses were examined. The a674r gene was cloned, and the protein was expressed in Escherichia coli. Biochemical characterization of the P. bursaria chlorella virus-1 recombinant ThyX protein indicates that it is more efficient at converting dUMP to dTMP than previously studied ThyX enzymes, thus allowing more detailed mechanistic studies of the enzyme. The ThyX-dUMP complexes with bound FAD function as efficient NAD(P)H oxidases, indicating that dUMP binds to the enzyme prior to NAD(P)H. This oxidation activity is directly linked to FAD reduction. Our results indicate that ThyX-specific inhibitors can be designed that do not affect ThyA enzymes. Finally, a model is proposed for the early stages of ThyX catalysis.
Laboratory of Optics and Biosciences, INSERM U451-CNRS UMR 7645, Ecole Polytechnique, 91128 Palaiseau Cedex, France.
Sébastien
S
Graziani
Yuannan
Y
Xia
James R
JR
Gurnon
James L
JL
Van Etten
Damien
D
Leduc
Stéphane
S
Skouloubris
Hannu
H
Myllykallio
Ursula
U
Liebl
D000818:Animals; D002708:Chlorella / Q000821:virology*; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000494:pharmacology*; D015870:Gene Expression; D009693:Nucleic Acid Hybridization; D010084:Oxidation-Reduction; D010247:Paramecium / Q000821:virology*; D017862:Phycodnaviridae / Q000201:enzymology* / Q000502:physiology; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D013938:Thymidine Monophosphate / Q000378:metabolism; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic; D014779:Virus Replication
D004279:DNA, Viral; D003856:Deoxyuracil Nucleotides; D004791:Enzyme Inhibitors; D005182:Flavin-Adenine Dinucleotide; D013938:Thymidine Monophosphate; D012313:RNA; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase