Functional Analysis of FAD-dependent Thymidylate Synthase ThyX from Paramecium bursaria Chlorella Virus-1

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 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...

Folate binding site of flavin-dependent thymidylate synthase

“…This suggestion is also consistent with the successful docking of NADPH into the newly identified folate binding site (Fig. S8) and with the CH 2 H 4 folate substrate-inhibition reported in the past (15,19). The present structure also shows a secondary and weaker (as evident form lower electron density) binding site for CH 2 H 4 folate at the surface of the protein (light yellow in Fig.…”

“…This observation led to the identification of alternative flavin-dependent thymidylate synthases (FDTSs), which are encoded by the thyX gene and have no sequence or structure homology to classic TSase enzymes (2,6,7). Furthermore, multiple studies have identified key differences in the molecular mechanism of catalysis between FDTSs and classic TSases (2,4,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). These differences, along with the fact that the thyX gene is present in many human pathogens (e.g., bacteria causing Anthrax, Tuberculosis, Typhus, and other diseases), renders these flavo-enzymes as potential targets for rational inhibitor design, possibly affording compounds that might be effective antimicrobial drugs (11,(22)(23)(24).…”

“…1A) from CH 2 H 4 folate to dUMP (4,8,10,(13)(14)(15)(18)(19)(20), or transfer of the methylene via an arginine residue (Arg174 in Thermatoga maritima FDTS; TmFDTS) before its delivery to dUMP (Fig. 1B) (16).…”

Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

“…This observation is analogous to a similar observation by Leduc et al (17) indicating that at least one serine residue in the vicinity of the ThyX motif is required for H. pylori ThyX catalysis. The double mutant data shown above support the essentiality of at least one histidine residue in the ThyX motif, as demonstrated in previous work (11,17). The R97 residue forms two hydrogen bonds to FAD and is highly conserved among ThyX proteins, and the R97A mutant protein did not complement growth.…”

Chlorella Virus-Encoded Deoxyuridine Triphosphatases Exhibit Different Temperature Optima

“…Instead, PBCV-1 encodes a flavindependent enzyme, called thymidylate synthase X, that synthesizes thymidylate (31). The recombinant PBCV-1 thymidylate synthase X is active (16), creating a PBCV-1 encoded pathway for dTMP synthesis that is part of dTTP biosynthesis (Fig. 7).…”