Although deoxythymidylate cannot be provided directly by ribonucleotide reductase, the gene encoding thymidylate synthase ThyA is absent from the genomes of a large number of nonsymbiotic microbes. We show that ThyX (Thy1) proteins of previously unknown function form a large and distinct class of thymidylate synthases. ThyX has a wide but sporadic phylogenetic distribution, almost exclusively limited to microbial genomes lacking thyA. ThyX and ThyA use different reductive mechanisms, because ThyX activity is dependent on reduced flavin nucleotides. Our findings reveal complexity in the evolution of thymidine in present-day DNA. Because ThyX proteins are found in many pathogenic microbes, they present a previously uncharacterized target for antimicrobial compounds.
SummaryFur, the ferric uptake regulator, is a transcription factor that controls iron metabolism in bacteria. Binding of ferrous iron to Fur triggers a conformational change that activates the protein for binding to specific DNA sequences named Fur boxes. In Helicobacter pylori, HpFur is involved in acid response and is important for gastric colonization in model animals. Here we present the crystal structure of a functionally active HpFur mutant (HpFur2M; C78S-C150S) bound to zinc. Although its fold is similar to that of other Fur and Fur-like proteins, the crystal structure of HpFur reveals a unique structured N-terminal extension and an unusual C-terminal helix. The structure also shows three metal binding sites: S1 the structural ZnS 4 site previously characterized biochemically in HpFur and the two zinc sites identified in other Fur proteins. Site-directed mutagenesis and spectroscopy analyses of purified wild-type HpFur and various mutants show that the two metal binding sites common to other Fur proteins can be also metallated by cobalt. DNA protection and circular dichroism experiments demonstrate that, while these two sites influence the affinity of HpFur for DNA, only one is absolutely required for DNA binding and could be responsible for the conformational changes of Fur upon metal binding while the other is a secondary site.
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.
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