Catalytic Mechanism and Structure of Viral Flavin-dependent Thymidylate Synthase ThyX

Abstract

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...

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

“…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. 1B) (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 (10,16,27). Furthermore, dUMP has been cocrystalized with T. maritima and M. tuberculosis ThyX (20) and NADPH was observed in the active site of M. tuberculosis ThyX (27); however, the position of the methylenetetrahydrofolate within the enzyme is not known.…”

“…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 (1,10,17,22,23). 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).…”

“…The structures of the ThyX protein from Thermotoga maritima (16), M. tuberculosis (27), and Paramecium bursaria chlorella virus-1 (10) have been solved recently, and the basic structures are very similar. All three enzymes are homotetramers, and they are structurally and evolutionarily unrelated to any of the ThyA enzymes that have been studied (22,23).…”

Folate binding site of flavin-dependent thymidylate synthase

“…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).…”