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.

Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase

“…It is worth noting that there is no evidence in the NMR spectrum of the reduced enzyme complex for rapid chemical exchange between uracil and dihydrouracil, which would be expected if the uracil moiety were rapidly reduced and re-oxidized by the flavin as invoked recently in a mechanism that proposed hydride transfer to C6 activated uracil as a nucleophile. 4 …”

An unprecedented mechanism of nucleotide methylation in organisms containing thyX

“…All FDTS mechanisms proposed heretofore ( 6-9 ) (e.g., Fig. S3) postulate that the methylene is transferred directly from CH 2 H 4 fol to the nucleotide, as in the TSase reaction ( 10 ), and the absorbance spectrum of the trapped intermediate (Fig.…”

Flavin-dependent thymidylate synthase: N5 of flavin as a Methylene carrier

“…In addition, the oxidation of flavin takes place slightly before the product starts to accumulate (Fig. 4A, green trace) [43]. …”

“…4A) can be explained via either of the mechanisms c or d (Scheme 3). In order to distinguish between mechanisms c and d, acid quenching was repeated in D 2 O (>99.9%) [43] with the expectation of trapping singly deuterated 5-HM-dUMP (one mass unit heavier) if mechanism c was taking place. On the other hand, if mechanism d is operative, then the intermediate trapped in D 2 O would not be heavier than the intermediate trapped in H 2 O.…”

“…Inhibitors with high specificity for FDTS over TSase are sought, especially in low nanomolar K i range, which is something that may be aided by the fact that FDTS is the only known uracil-methylating enzyme without a catalytic nucleophile [28,43]. Substrate analogues have been examined for this endeavor, and crystal structures of Tm FDTS with a variety of possible inhibitors have been obtained.…”