Substrate activation in flavin-dependent thymidylate synthase

Abstract

Thymidylate is a critical DNA nucleotide that has to be synthesized in cells de novo by all organisms. Flavin-dependent thymidylate synthase (FDTS) catalyzes the final step in this de novo production of thymidylate in many human pathogens, but it is absent from humans. The FDTS reaction proceeds via a chemical route that is different from its human enzyme analogue, making FDTS a potential antimicrobial target. The chemical mechanism of FDTS is still not understood, and the two most recently proposed mechanisms involve reaction intermediates that are unusual in pyrimidine biosynthesis and biology in general. These mechanisms differ in the relative timing of the reaction of the flavin with the substrate. The consequence of this difference is significant: the intermediates are cationic in one case and neutral in the other, an important consideration in the construction of mechanism-based enzyme inhibitors. Here we test these mechanisms via chemical trapping of reaction intermediates, stopped-flow, and substrate hydrogen isotope exchange techniques. Our findings suggest that an initial activation of the pyrimidine substrate by reduced flavin is required for catalysis, and a revised mechanism is proposed on the basis of previous and new data. These findings and the newly proposed mechanism add an important piece to the puzzle of the mechanism of FDTS and suggest a new class of intermediates that, in the future, may serve as targets for mechanism-based design of FDTS-specific inhibitors.

Scheme 1. Proposed Chemical Mechanisms for FDTS

Adapted with permission from ref (11). R = 2′-deoxyribose-5′-phosphate; R′ = (p-aminobenzoyl)glutamate; R″ = adenosine-5′-pyrophosphate-ribityl.

Figure 1

Single-turnover FDTS reaction kinetics overlaid with stopped-flow flavin absorbance trace (green, this work). Reduced flavin (FADH₂) has no 420 nm absorbance, while oxidized flavin (FAD) does. Adapted with permission from ref (11).

Scheme 2. Acid Trapping of the Proposed Intermediates in the Reaction with Deuterium-Labeled Flavin (FADD₂)

Formation of 5-HM-dUMP in (a) requires oxidation of the reduced intermediates at C6, i.e., loss of a hydron (H⁺ or D⁺) and two electrons. Due to an isotope effect on this nonenzymatic oxidation, the majority of 5-HM-dUMP is expected to be deuterated. Molecular oxygen has been proposed as the oxidant, since quenched reactions are exposed to oxygen during quenching.

Figure 2

HRMS of 5-hydroxymethyl-dUMP isolated from the acid-quenched FDTS reactions in H₂O and D₂O.

Figure 3

ESI-MS spectra of dUMP incubated in D₂O with dithionite (a), dithionite-reduced FDTS (b), oxidized FDTS (c), and NADPH-reduced FDTS (d). All spectra were collected in the negative-ion mode.

Scheme 3. Proposed Alternative Mechanism for FDTS That Agrees with Both Current and Past Findings

The hypothesis is that steps 1–3 occur within the dead-time of the flow experiments (2 ms), and that intermediates between steps 3 and 5 accumulate and are trapped by the acid in the quench-flow experiment. At this time it is not clear if the elimination of H₄fol precedes the hydride transfer from the flavin (step 4a) or is concerted with it (the green arrows in step 4b). Note that FAD prosthetic group remains bound to the enzyme throughout the catalytic cycle, although its isoalloxazine ring fluctuates toward and away from the substrate as described in ref (21).