Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase

Abstract

Many microorganisms use flavin-dependent thymidylate synthase (FDTS) to synthesize the essential nucleotide 2'-deoxythymidine 5'-monophosphate (dTMP) from 2'-deoxyuridine 5'-monophosphate (dUMP), 5,10-methylenetetrahydrofolate (CH2THF), and NADPH. FDTSs have a structure that is unrelated to the thymidylate synthase used by humans and a very different mechanism. Here we report nuclear magnetic resonance evidence that FDTS ionizes N3 of dUMP using an active-site arginine. The ionized form of dUMP is largely responsible for the changes in the flavin absorbance spectrum of FDTS upon dUMP binding. dUMP analogues also suggest that the phosphate of dUMP acts as the base that removes the proton from C5 of the dUMP-methylene intermediate in the FDTS-catalyzed reaction. These findings establish additional differences between the mechanisms of FDTS and human thymidylate synthase.

Figure 1

Thymidylate synthase chemical mechanisms. (a) the mechanism for classic TS using an enzymatic cysteine-nucleophile. (b) a proposed mechanism for FDTS using an electrostatically polarizing active site to activate dUMP.

Figure 2

Active site of flavin-dependent thymidylate synthase (PDB code: 1O26). In the mechanism shown in Figure 1B, the arrangement of R90, R174, and the phosphate of dUMP (cyan) are proposed to stabilize a resonance form of dUMP with enhanced nucleophilicity at C5. Also, note the close proximity of the phosphate oxygens to C5 of the uracil.

Figure 3

Spectrophotometric titrations of WT (A) and R174A (B) FDTS with dUMP in 0.1 M Tris-HCl, pH 8 at 25 °C. The insets show the change in absorbance as a function of ligand concentration. The spectral change for dUMP binding to R174A FDTS was noticeably different from other FDTS variants. Fitting to Eq. 1 gives a Kd of 30 nM for dUMP binding to WT FDTS. Fitting to a square hyperbola gives a Kd of 109 μM for dUMP binding to R174A FDTS.

Figure 4

The ¹³C-NMR spectra of uracil carbons of dUMP/dU free in solution or bound to WT and variant FDTSs at 45 °C. All of the ligand-FDTS complexes were at pH 8. The C4 and C2 signals for dUMP shifted upfield ~8 ppm when bound to WT FDTS, having nearly identical chemical shifts as free dUMP at pH 12. Mutagenesis of R174 – which is 2.8 Å from N3 of dUMP in the dUMP-WT FDTS structure – to alanine eliminated the changes in chemical shifts of the dUMP-FDTS complex. In contrast, mutagenesis of R90 to alanine or removal of the phosphate of dUMP had little effect on the ¹³C-NMR spectrum of the uracil carbons in the complex. Highlighted in maize and blue, respectively, are the chemical shifts of the carbonyl carbons of N3-ionized and N3-unionized uracil.

Figure 5

Proton expulsion upon dUMP binding. Phenol red was added to unbuffered FDTS (10 μM in FAD) in 50 mM NaCl, pH 8.0. Aliquots of concentrated dUMP at pH 8.0 was added and spectra were recorded. Proton release upon dUMP binding was evident by the decrease in absorbance of deprotonated phenol red. The inset shows that proton releases ceases at ~1:1 stoichiometry of dUMP:FAD.

Figure 6

Overlay of WT and variant structures. dUMP/dU/dUMS bind in the same position and orientation in all of the active site variants used in this study. dUMP + WT (PDB code: 1O26), blue; dU + WT, green; dUMP + R90A, gray; dUMP + R174A, magenta; dUMS + WT, maize.

Figure 7

dU failed to oxidize reduced FDTS in the presence of CH2THF. Anaerobic oxidized FDTS (green) in 0.1 M Tris-HCl, pH 8 at 25 °C was titrated stoichiometrically with dithionite to reduce the flavin (blue). Anaerobic addition of CH2THF perturbed the spectrum, indicating that it formed a complex with the enzyme (black). Anaerobic addition of dU to the reduced FDTS-CH2THF complex perturbed the spectrum further but failed to oxidize the flavin (orange), indicating that the phosphate of dUMP plays an essential role in the FDTS-catalyzed reaction. The flavin could then be re-oxidized by air (red).

Figure 8

Binding and reactivity of dUMS in 0.1 M Tris-HCl, pH 8 at 25 °C. A, spectrophotometric titration of WT FDTS with dUMS. dUMS produced a flavin spectral change similar to that produced by dUMP. Fitting to Eq. 1 gave a Kd of 170 nM. B, dUMS failed to oxidize reduced FDTS in the presence of CH2THF. Anaerobic oxidized FDTS (green) was titrated stoichiometrically with dithionite to reduce the flavin (blue). Anaerobic addition of CH2THF perturbed the spectrum of the flavin (black), indicating that it formed a complex with the enzyme. Anaerobic addition of dUMS to the reduced FDTS-CH2THF complex also perturbed the spectrum (orange), indicating binding, but it failed to oxidize the enzyme. The flavin was then re-oxidized by exposure to air (red).

Figure 9

Proposed mechanism for the reaction of FDTS. dUMP is activated by deprotonating N3 (1). The negative charge delocalizes onto O4 and O2, isolating electronically the enamine substructure (highlighted in yellow), enhancing its nucleophilicity for reaction with the methylene carbon of the flavin-iminium adduct proposed. Removal of the C5 proton of the bridged adduct by the phosphate of dUMP leads, ultimately, to production of dTMP and oxidized FAD.