TY - JOUR
T1 - Functional analysis of FAD-dependent thymidylate synthase ThyX from Paramecium bursaria chlorella virus-1
AU - Graziani, Sébastien
AU - Xia, Yuannan
AU - Gurnon, James R.
AU - Van Etten, James L.
AU - Leduc, Damien
AU - Skouloubris, Stéphane
AU - Myllykallio, Hannu
AU - Liebl, Ursula
N1 - Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2004/12/24
Y1 - 2004/12/24
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.M409121200
DO - 10.1074/jbc.M409121200
M3 - Article
C2 - 15471872
AN - SCOPUS:11144235173
VL - 279
SP - 54340
EP - 54347
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 52
ER -