By using biochemical and structural analyses, we have investigated the catalytic mechanism of the recently discovered flavindependent 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 (His 53 ) 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.All cellular organisms need thymidylate (dTMP) for the replication of their chromosomes, as dTMP is required for the biosynthesis of dTTP, a building block of DNA. Cells can produce thymidylate either de novo from dUMP or incorporate thymidine using thymidine kinase. The de novo pathway of dTMP synthesis requires a specific enzyme, thymidylate synthase, that methylates dUMP at position 5 of the pyrimidine ring. Two structurally and mechanistically distinct classes of thymidylate synthases exist. The well studied ThyA proteins (EC 2.1.1.45) catalyze the reductive methylation reaction of dUMP, with methylenetetrahydrofolate (CH 2 H 4 folate) 6 serving as one-carbon donor and as source of reductive power (reviewed in Ref. 1).On the other hand, the recently discovered ThyX (EC 2.1.1.148) family of thymidylate synthases contains FAD (2) that is tightly bound by a novel fold (3). FAD mediates hydride transfer from NADPH during catalysis (4 -6). Consequently, in the reaction catalyzed by ThyX, CH 2 H 4 folate serves only as a carbon donor, leading to the prediction that tetrahydrofolate (and not dihydrofolate as is the case for ThyA) is produced (2). This prediction has recently been confirmed by identifying tetrahydrofolate as a reaction product of Chlamydia trachomatis ThyX using high pressure liquid chromatography (7).The catalytic reaction of thymidylate synthase ThyA is a sequential ordered mechanism in which dUMP binding is followed by the entry of CH 2 H 4 folate, and subsequent ternary complex formation with dUMP and CH 2 H 4 folate simultaneously bound to the enzyme (8, 9). This was demonstrated by thorough steady-state kinetic measurements using varying concentrations of these two substrates of the ThyA reaction. Moreover, by using fluoro-dUMP in the reaction mixtures, this covalent ternary complex can readily be trapped for ThyA proteins (10). Although ThyX catalysis is of considerable interest for detecting and designing new anti-microbial compounds (2), our understanding of the reaction mechanism of this enzyme is still incomplete. Several models propose that the cata...

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. Paramecium bursaria chlorella virus-1 (PBCV-1)1 is a large double-stranded DNA virus that replicates in certain unicellular eukaryotic chlorella-like green algae (1). By 4 h after infection, the DNA concentration in a virus-infected cell increases 4 -10-fold because of viral DNA synthesis (2). Viral DNA synthesis presumably requires higher concentrations of deoxynucleotides (dNTPs) than the host can supply, implying that large quantities of dNTPs need to be synthesized de novo by viral encoded proteins. Genome sequencing of PBCV-1 revealed that the virus encodes at least 13 putative enzymes involved in DNA precursor metabolism (1), among them dUTP pyrophosphatase and dCMP deaminase that participate in the formation of dUMP from dUTP and dCMP, respectively. dUMP is a substrate for thymidylate synthase and is required for de novo synthesis of thymidylate (dTMP), an essential DNA precursor. Whereas PBCV-1 lacks a canonical thymidylate synthase ThyA (EC 2.1.1.45), its open reading frame A674R has a highly conserved sequence motif RHRX 7 S ("ThyX motif") as well as significant overall amino acid sequence similarity to an alternative class of thymidylate synthases called ThyX (EC 2.1.1.148) (3). ThyX proteins are found in many pathogenic bacteria and several double-stranded DNA viruses. Although numerous ThyA homologs have been analyzed from viral sources, no data are available for viral ThyX proteins.The homodimeric ThyA (4) and homotetrameric ThyX proteins (3, 5) have no sequence or structural similarity, but both catalyze the methylation of dUMP to dTMP. Although both ThyA and ThyX depend on methylenetet...

Little is known about the catalytic mechanism of the recently discovered ThyX family of flavin-dependent thymidylate synthases that are required for thymidylate (deoxythymidine 5 -monophosphate) synthesis in a large number of microbial species. Using a combination of site-directed mutagenesis and biochemical measurements, we have identified several residues of the Helicobacter pylori ThyX protein with crucial roles in ThyX catalysis. By providing functional evidence that the active site(s) of homotetrameric ThyX proteins is formed by three different subunits, our findings suggest that ThyX proteins have evolved through multimerization of inactive monomers. Moreover, because the active-site configurations of ThyX proteins, present in many human pathogenic bacteria, and of human thymidylate synthase ThyA are different, our results will aid in the identification of compounds specifically inhibiting microbial growth.

RNA polymerase (Pol) III synthesizes the tRNAs, the 5S ribosomal RNA and a small number of untranslated RNAs. In vitro, it also transcribes short interspersed nuclear elements (SINEs). We investigated the distribution of Pol III and its associated transcription factors on the genome of mouse embryonic stem cells using a highly specific tandem ChIP-Seq method. Only a subset of the annotated class III genes was bound and thus transcribed. A few hundred SINEs were associated with the Pol III transcription machinery. We observed that Pol III and its transcription factors were present at 30 unannotated sites on the mouse genome, only one of which was conserved in human. An RNA was associated with >80% of these regions. More than 2200 regions bound by TFIIIC transcription factor were devoid of Pol III. These sites were associated with cohesins and often located close to CTCF-binding sites, suggesting that TFIIIC might cooperate with these factors to organize the chromatin. We also investigated the genome-wide distribution of the ubiquitous TFIIS variant, TCEA1. We found that, as in Saccharomyces cerevisiae, TFIIS is associated with class III genes and also with SINEs suggesting that TFIIS is a Pol III transcription factor in mammals.

The hyperthermophilic anaerobic archaeon Pyrococcus abyssi, which lacks thymidine kinase, incorporates label from extracellular uracil, but not from thymidine, into its DNA. This implies that P. abyssi must synthesize dTMP (thymidylate), an essential precursor for DNA synthesis, de novo. However, iterative similarity searches of the three completed Pyrococcus genomes fail to detect candidate genes for canonical thymidylate synthase ThyA, suggesting the presence of alternative pathways for dTMP synthesis. Indeed, by identifying a novel class of flavin-dependent thymidylate synthases, ThyX, we have recently proven that two distinct pathways for de novo synthesis of dTMP are operational in the microbial world. While both thyX and thyA can be found in hyperthermophilic micro-organisms, the phylogenetic distribution of thyX among hyperthermophiles is wider than that of thyA. In this contribution, we discuss the differences in the distinct mechanisms of dTMP synthesis, with a special emphasis on hyperthermophilic micro-organisms.

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