An unprecedented mechanism of nucleotide methylation in organisms containing thyX

Abstract

In several human pathogens, thyX-encoded flavin-dependent thymidylate synthase (FDTS) catalyzes the last step in the biosynthesis of thymidylate, one of the four DNA nucleotides. ThyX is absent in humans, rendering FDTS an attractive antibiotic target; however, the lack of mechanistic understanding prohibits mechanism-based drug design. Here, we report trapping and characterization of two consecutive intermediates, which together with previous crystal structures indicate that the enzyme's reduced flavin relays a methylene from the folate carrier to the nucleotide acceptor. Furthermore, these results corroborate an unprecedented activation of the nucleotide that involves no covalent modification but only electrostatic polarization by the enzyme's active site. These findings indicate a mechanism that is very different from thymidylate biosynthesis in humans, underscoring the promise of FDTS as an antibiotic target.

Fig. 1

Reactions catalyzed by TSase, DHFR, and FDTS. Highlighted are the reducing hydrogen in the TSase reaction (red), methylene (magenta), and nucleotide under study (blue). R = 2'-deoxyribose-5'-phosphate; R' = (p-aminobenzoyl)glutamate.

Fig. 2

Oxidative half-reaction kinetics of FDTS. (Top panel) Base-quenched data (dots) globally fitted to a single-intermediate model (lines), overlaid with stopped-flow trace at 420 nm (flavin absorbance in green) (). Each time point was obtained from a radiogram like that shown in Fig. S1A, with dUMP in red, dTMP in black, and intermediate in blue. (Bottom panel) Base-trapped intermediate kinetics (blue) overlaid with acid-trapped intermediate data (red), globally fitted to a two-intermediate model (red curves). The combined total of the two intermediates is shown as a dashed red curve.

Fig. 3

Structure of the base-modified FDTS reaction intermediate. (Top panel) Structural components originating in dUMP (red) and CH₂H₄fol (black) are referred to as “U subunit” and the structural parts derived from FAD (blue) as “F subunit.” Atomic numbering in F subunit follows convention adapted from the original FAD (Fig. S5A). (Bottom panel) Overlay of ¹H/¹³C HMQC (black; red marks folded cross peaks) and HMBC (green) spectra. The cross peaks are labeled with the first letter representing the subunit (U or F). The cross peaks of the HMBC spectrum circled in red unambiguously establish that the CH₂H₄fol-drived methylene (U7) bridges dUMP and flavin derivative.

Fig. 4

Proposed chemical mechanism for FDTS. (Left panel) Methylene (CH_2, magenta) is transferred from the N5 of H₄fol (green) via N5 of FAD cofactor (gold) to C5 of dUMP substrate (cyan). Oxygens are in red, nitrogens in blue, and the hydride transferred in step 4 in brown. The proposed steps of flavin oxidative half-reaction are numbered. I₁ (boxed) is the reaction intermediate that in base is converted to the compound presented in Fig. 3, and in acid undergoes water addition (Fig. S10 and Supplementary Discussion). I₂ (boxed) refers to the intermediate that in base forms dTMP, and in acid reacts with water (Fig. S10 and Supplementary Discussion). The “420 nm absorbance” vs. “colorless” labels of flavins relate the 420 nm time-trace (green line in Fig. 3) to the mechanistic model. A rigorous electron-pushing description of this mechanism is presented in Fig. S8. R = 2′-deoxyribose-5′-phosphate, R′ = (p-aminobenzoyl)glutamate, and R"′ = adenosine-5′-pyrophosphate–ribityl. (Right panel) Active site of FDTS (PDB ID 4GTA ()) in complex with FAD, dUMP, and folinic acid, a stable analogue of the activated CH₂H₄fol. For clarity some the carbonyl oxygen of folinic acid are not shown, and the structure is inversed to match the orientation of ligands in the left panel. The formyl carbon (magenta) on folinic acid represents the methylene to be transferred. Black arrows mark the methylene transfer trajectory from donor (N5 of folate), to proposed relay atom (N5 of flavin), and final acceptor (C5 of dUMP).