Functional analysis of FAD-dependent thymidylate synthase ThyX from Paramecium bursaria chlorella virus-1

Sébastien Graziani, Yuannan Xia, James R. Gurnon, James L. Van Etten, Damien Leduc, Stéphane Skouloubris, Hannu Myllykallio, Ursula Liebl

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56 Scopus citations

Abstract

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 methylenetetraliydrofolate (CH2H 4folate) as both a source of the methylene group and the reductant, CH2H4folate 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.

Original languageEnglish (US)
Pages (from-to)54340-54347
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number52
DOIs
StatePublished - Dec 24 2004

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Graziani, S., Xia, Y., Gurnon, J. R., Van Etten, J. L., Leduc, D., Skouloubris, S., Myllykallio, H., & Liebl, U. (2004). Functional analysis of FAD-dependent thymidylate synthase ThyX from Paramecium bursaria chlorella virus-1. Journal of Biological Chemistry, 279(52), 54340-54347. https://doi.org/10.1074/jbc.M409121200