The relationships between oxidase and synthase activities of flavin dependent thymidylate synthase (FDTS)

Abstract

New findings lead to a revised understanding of the substrates' binding order, the role of the substrate as an activator, and the observed lag phase in the FDTS catalyzed reaction.

Folate binding site of flavin-dependent thymidylate synthase

“…To test this hypothesis, we examined the effect of folate moieties on the single reductive-half reaction of FAD with NADPH. Steady-state competitive CH 2 H 4 folate inhibition has been reported for the oxidase activity of FDTS (10,14), indicating that NADPH and folate cannot both be bound to the active site at the same time, thus supporting the current suggestion. Nevertheless, such competitive inhibition under steady-state conditions could be complicated by the reported positive cooperativity (15), and the possible allosteric binding site (light yellow in Fig.…”

“…1B) (16). These studies may have been complicated by the competing oxidase activity (FADH 2 reaction with O 2 to form H 2 O 2 ) (10,14,27) and by other kinetic complexities, such as possible positive cooperativity (15). Published structures of FDTSs (7,9,15,29,30) have not been reported with folates, making further differentiation between these methylene transfer mechanisms difficult.…”

“…The FAD can be reduced to FADH 2 by nicotinamides, ferrodoxin, and other small molecule reductants (e.g., dithionite). Although the reductive halfreaction is activated by dUMP the conversion of dUMP to dTMP occurs during the oxidative half-reaction (FADH 2 →FAD) (8,10,14,18,27). In contrast to classic TSase, where the cofactor CH 2 H 4 folate provides both the H − and methylene, in the FDTS reaction the H − is provided by the FADH 2 and CH 2 H 4 folate is used only as a source for the methylene moiety (18,20,28).…”

“…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).…”