Thymidine biosynthesis is essential in virtually all organisms, but two different pathways have evolved to accomplish this critical task. The two thymidylate synthesis enzymes ThyA and ThyX are structurally and evolutionarily unrelated (16
), 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 ThyA-deficient E. coli
). ThyA is used commonly in eukaryotes and in many microorganisms, including most eubacteria. ThyX has been identified more recently, and it is common among the archaea and some eubacteria, particularly those that are anaerobic or occupy extreme environments. The corynebacteria, the group that includes the mycobacteria, are unusual in having both ThyA and ThyX in their genomes. The extreme conservation of the thyX
sequences within this group suggests that both genes serve essential functions for these organisms, but the roles for two parallel pathways are not yet defined. This study of the key residues in the M. tuberculosis
ThyX enzyme is the first step toward understanding the function of this enzyme in the growth and various physiological states of this pathogen in its active and latent phases.
The classic pathway uses the ThyA enzyme to methylate dUMP in the final step of thymidine biosynthesis (3
). In the alternative pathway, ThyX performs the transfer without oxidation of tetrahydrofolate, utilizing both dUMP and methylenetetrahydrofolate as reactants and FAD and NADPH in the redox phase of the reaction (1
). Whereas ThyA requires a companion enzyme, dihydrofolate reductase, to regenerate tetrahydrofolate that has been oxidized in the transfer reaction, ThyX requires dUMP, NADPH, FAD, and methylenetetrahydrofolate (3
). Currently, four reaction mechanisms of ThyX have been proposed, as shown in Fig. (1
). Our data have identified mutants (e.g., the Y44F, Y108S, V109E, R190A, F195V, and R199A mutants) that are still able to oxidize NADPH but are unable to catalyze the deprotonation reaction. This behavior is consistent with NADPH oxidation taking place before deprotonation of dUMP, as shown in Fig. . Therefore, these analyses allowed identification of several residues that normally participate in coupling the NADPH oxidation and deprotonation reactions of ThyX. Some of these amino acid residues have interactions with FAD and/or dUMP, as shown in the crystallographic image of the M. tuberculosis
ThyX active site (see Fig. S1 in the supplemental material).
We focused this study on amino acids that had been identified in two ways. First, alignment of the many eubacterial and archaeal thyX genes showed a set of highly conserved residues; these had been used to define a ThyX signature motif, RHRX7-8S. The structure of the M. tuberculosis ThyX protein was solved with both FAD and BrdUMP bound, and this structure detailed important interactions of the protein and these molecules in crystals. Our goal was to test the validity of the ThyX motif for the M. tuberculosis enzyme and the importance of the molecular interactions observed in crystals.
A number of interesting mutants provided more guidance on the details of the enzyme activity. For example, several substitutions of amino acid residues with similar side groups successfully complemented the E. coli
χ2913 strain (E74Q, S100T, Y108F, and T181S) whereas the less conservative substitutions failed to complement (E74A, S100A, Y108S, and T181L). Moreover, there were mutants that failed to complement the E. coli
χ2913 strain that did catalyze the NADPH oxidation reaction in vitro (the Y44F, Y108S, F195V, and R199A mutants). There were no examples of mutants that catalyzed the 3
H release but not the oxidation. The simplest explanation for this asymmetry is that the oxidation step of the reaction precedes deprotonation, which provides further support to the reaction mechanisms wherein NADPH oxidation precedes deprotonation (Fig. ) (10
When the structures of T. maritima
, M. tuberculosis
, and P. bursaria
chlorella virus-1 ThyX were solved, FAD was identified as being firmly bound within these ThyX enzymes (10
). Furthermore, dUMP has been cocrystalized with T. maritima
and M. tuberculosis
) and NADPH was observed in the active site of M. tuberculosis
); however, the position of the methylenetetrahydrofolate within the enzyme is not known. Under low-pH conditions (pH 4.5 to 5.5), NADPH was observed to displace FAD from preformed crystals in M. tuberculosis
). Nevertheless, the fact that two of the residues (R190 and H194) that interacted with NADPH in the crystals also interacted with tightly bound FAD in the crystals (26
) indicates that the location of NADPH binding might not be biologically relevant. Two mutant proteins (the S100A and L104A mutants) were able to catalyze both the oxidation and the 3
H release in vitro but still failed to complement the E. coli
χ2913 strain. This failure to complement is probably due to a defect in a later stage of the reaction possibly involving the transfer of C1
units or the release of dTMP. These data provide the first clues to residues essential for the final steps of methylene transfer to the dUMP and/or release of the tetrahydrofolate.
In ThyA enzymes, a cysteine residue acts as the nucleophile by covalently binding the position 6-carbon of dUMP and stabilizing the deoxyribose ring (3
). In all ThyX enzymes sequenced to date, a serine residue appears to function as the nucleophile and forms an integral part of the ThyX motif (see Table S2 in the supplemental material). The S105C mutant was unable to function as the nucleophile in ThyX, and this further underscores the fundamental differences between the two thymidylate synthase enzymes.
Several positions in the X7-8 portion of the previously defined ThyX motif are not highly conserved among the thyX genes so far identified. It is likely that these serve to position key neighboring residues for their enzymatic function.
The previously defined motif was defined principally by alignment of the thyX genes so far identified (see Table S2 in the supplemental material), and our mutagenesis study has added to our understanding of the role of particular amino acids in the enzyme activity. Despite our work, it is still unclear which residues contribute to binding of methylenetetrahydrofolate and NADPH. Nonetheless, the combination of structural and mutagenesis studies reported here has provided a clearer picture of FAD and dUMP binding residues. Our analyses permit us to propose an extended range of the ThyX motif, shown here with the underlined histidine acting as the catalytic residue and the underlined serine as the nucleophile: (Y/W)X19-40HX25-28RHRX7-8SXR(Y/F)X68-114R.
The strong evolutionary conservation of the thyX
gene suggests that our study of the M. tuberculosis
enzyme will be useful for analysis of this enzyme with a wide range of human pathogens, including those that depend entirely on ThyX for biosynthesis of thymidine. Since this reaction is clearly essential in those organisms, the identification of inhibitors is a first step in developing drugs effective against them. When ThyX inhibitors are available, this work will aid in understanding their inhibitory effects. Because mycobacteria have both ThyA and ThyX pathways for thymidine synthesis, any effective drug might need to inhibit both enzymes. However, ThyA requires concomitant dihydrofolate reductase to regenerate tetrahydrofolate, so a combination of ThyX and DHFR inhibition might limit M. tuberculosis
growth. Promising inhibitors of M. tuberculosis
DHFR have been identified previously (7
). Our detailed determination of the function of key residues in the ThyX enzyme should facilitate further study of this potential drug target and will help to better understand the complex reactions of this interesting enzyme.