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.

The gene for a reaction center core polypeptide from the anoxygenic photosynthetic bacterium Heliobacillus mobilis was cloned and sequenced. The deduced amino acid sequence consists of 609 residues with a molecular mass of 68 kDa. An adjacent open reading frame is not transcribed under our experimental conditions. No evidence for a second related reaction center core gene was found. The primary sequence of the reaction center protein (P800 protein) shows a high percentage of sequence identity to photosystem I in a cysteine-containing loop, which is the putative binding site of the iron-sulfur center FX and in the preceding hydrophobic region. Our data imply a homodimeric organization of the reaction center. This is fundamentally different from photosystem I and most other photosynthetic reaction centers, where the reaction center core is composed of two similar but nonidentical subunits.

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...

The interaction of mitochondrial cytochrome (cyt) c with cardiolipin (CL) is involved in the initial stages of apoptosis. This interaction can lead to destabilization of the heme-Met80 bond and peroxidase activity [Basova, L. V., et al. (2007) Biochemistry 46, 3423-3434]. We show that under these conditions carbon monoxide (CO) binds to cyt c, with very high affinity ( approximately 5 x 10(7) M(-1)), in contrast to the native cyt c protein involved in respiratory electron shuttling that does not bind CO. Binding of CO to the cyt c-CL complex inhibits its peroxidase activity. Photodissociated CO from the cyt c-CL complex shows <20% picosecond geminate rebinding and predominantly bimolecular rebinding, with a second-order rate constant of approximately 10(7) M(-1) s(-1), an order of magnitude higher than in myoglobin. These findings contrast with those of Met80X mutant cyt c, where picosecond geminate recombination dominates due to the rigidity of the protein. Our data imply that CL leads to substantial changes in protein conformation and flexibility, allowing access of ligands to the heme. Together with the findings that (a) approximately 30 CL per cyt c are required for full CO binding and (b) salt-induced dissociation indicates that the two negative headgroup charges interact with approximately 5 positive surface charges of the protein, these results are consistent with a CL anchorage model with an acyl chain impaled in the protein [Kalanxhi, E., and Wallace, C. J. A. (2007) Biochem. J. 407, 179-187]. The affinity of CO for the complex is high enough to envisage an antiapoptotic effect of nanomolar CO concentrations via inhibition of the cyt c peroxidase activity.

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.

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...

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Heme-based oxygen sensors are part of ligand-specific twocomponent regulatory systems, which have both a relatively low oxygen affinity and a low oxygen-binding rate. To get insight into the dynamical aspects underlying these features and the ligand specificity of the signal transduction from the heme sensor domain, we used femtosecond spectroscopy to study ligand dynamics in the heme domains of the oxygen sensors FixL from Bradyrhizobium japonicum (FixLH) and Dos from Escherichia coli (DosH). The heme coordination with different ligands and the corresponding ground-state heme spectra of FixLH are similar to myoglobin (Mb). After photodissociation, the excited-state properties and ligand-rebinding kinetics are qualitatively similar for FixLH and Mb for CO and NO as ligands. In contrast to Mb, the transient spectra of FixLH after photodissociation of ligands are distorted compared with the ground-state difference spectra, indicating differences in the heme environment with respect to the unliganded state. This distortion is particularly marked for O 2. Strikingly, heme-O 2 recombination occurs with efficiency unprecedented for heme proteins, in Ϸ5 ps for Ϸ90% of the dissociated O2. For DosH-O2, which shows 60% sequence similarity to FixLH, but where signal detection and transmission presumably are quite different, a similarly fast recombination was found with an even higher yield. Altogether these results indicate that in these sensors the heme pocket acts as a ligand-specific trap. The general implications for the functioning of heme-based ligand sensors are discussed in the light of recent studies on heme-based NO and CO sensors.