An Alternative Flavin-Dependent Mechanism for Thymidylate Synthesis

Abstract

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.

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

“…The two thymidylate synthesis enzymes ThyA and ThyX are structurally and evolutionarily unrelated (16,22,23), but clearly they perform the same function. This similarity is illustrated most dramatically by the fact that the M. tuberculosis and H. pylori ThyX enzymes can both complement ThyAdeficient E. coli (17,27).…”

“…All three enzymes are homotetramers, and they are structurally and evolutionarily unrelated to any of the ThyA enzymes that have been studied (22,23). The genes that encode the putative ThyX enzyme have been identified in organisms from several hundred species, representing more than 70 genera, and there are some highly conserved amino acids that have been termed the ThyX motif (RHRX 7-8 S) (17,18).…”

“…ThyX in these organisms is an attractive drug target (4,6,15,23). Eubacterial species in the corynebacterium group are unusual because they have retained the genes for both the ThyA/ DHFR and the ThyX pathways (22,23). Within this group the sequences of the thyA genes are very highly conserved, and the thyX genes show almost complete identity (30).…”

“…Like most eukaryotes, humans depend upon ThyA, so there has been special interest in the enzyme of ThyX-dependent pathogens such as Helicobacter pylori, Campylobacter jejuni, Borrelia burgdorferi, and Treponema pallidum (see reference 22 for a review). ThyX in these organisms is an attractive drug target (4,6,15,23). Eubacterial species in the corynebacterium group are unusual because they have retained the genes for both the ThyA/ DHFR and the ThyX pathways (22,23).…”

“…For most of this time, research focused on the enzyme that is common in eukaryotes and many eubacteria, the thymidylate synthase enzyme ThyA (EC 2.1.1.45), which reductively methylates dUMP in tandem with dihydrofolate reductase DHFR (EC 1.5.1.3) (3). More recently, a novel FAD-dependent thymidylate synthase, ThyX (EC 2.1.1.148), which transfers the methyl group without oxidation of the tetrahydrofolate carrier, has been identified in a variety of eubacteria and archaea (9,22,23). Different from ThyA proteins, ThyX proteins use a redox-activated FAD cofactor to form dTMP (Fig.…”