Detection of Intermediates in the Oxidative Half-Reaction of the FAD-Dependent Thymidylate Synthase fromThermotoga maritima: Carbon Transfer without Covalent Pyrimidine Activation

Abstract

Thymidylate, a vital DNA precursor, is synthesized by thymidylate synthases (TSs). A second class of TSs, encoded by the thyX gene, is found in bacteria and a few other microbes and is especially widespread in anaerobes. TS encoded by thyX requires a flavin adenine dinucleotide prosthetic group for activity. In the oxidative half-reaction, the reduced flavin is oxidized by 2′-deoxyuridine 5′-monophosphate (dUMP) and (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2THF), synthesizing 2′-deoxythymidine 5′-monophosphate (dTMP). dTMP synthesis is a complex process, requiring the enzyme to promote carbon transfer, probably by increasing the nucleophilicity of dUMP and the electrophilicity of CH2THF, and reduction of the transferred carbon. The mechanism of the oxidative half-reaction was investigated by transient kinetics. Two intermediates were detected, the first by a change in the flavin absorbance spectrum in stopped-flow experiments and the second by the transient disappearance of deoxynucleotide in acid quenching experiments. The effects of substrate analogues and the behavior of mutated enzymes on these reactions lead to the conclusion that activation of dUMP does not occur through a Michael-like addition, the mechanism for the activation analogous with that of the flavin-independent TS. Rather, we propose that the nucleophilicity of dUMP is enhanced by electrostatic polarization upon binding to the active site. This conclusion rationalizes many of our observations, for instance, the markedly slower reactions when two arginine residues that hydrogen bond with the uracil moiety of dUMP were mutated to alanine. The activation of dUMP by polarization is consistent with the majority of the published data on ThyX and provides a testable mechanistic hypothesis.

Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase

“…A new mechanism for dUMP activation based on ionization of N3 follows from a modification of the mechanism 6 shown in Figure 1B. The negative charge of the ionized nitrogen produced when dUMP binds FDTS delocalizes onto O2 and O4 (Figure 9).…”

“…Recombinant WT and mutagenized FDTSs from T. maritima were purified as previously reported, 6 followed by formation of apo-protein by treatment with concentrated sodium chloride 10 and reincorporation of exogenous FAD after desalting.…”

“…1 (b) a proposed mechanism for FDTS using an electrostatically polarizing active site to activate dUMP. 6 …”

“…3 Hydride transfer from reduced FAD has been proposed to activate dUMP; 3,4 however, this is unlikely since 5-fluoro-2′-deoxyuridine-5′-monophosphate (5F-dUMP) cannot oxidize reduced FDTS in the presence of CH 2 THF, 5 and stopped-flow experiments on the oxidative half-reaction indicate multiple intermediates prior to flavin oxidation. 6 …”

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

“…Spectrophotometric studies have shown noticeable changes in the ultraviolet–visible (UV–vis) absorption spectra and the presence of reaction intermediates for the Tm FDTS catalyzed formation of dTMP (oxidative half-reaction; Fig. 3) [15,34,35]. Under anaerobic conditions, addition of either substrate, dUMP or CH 2 H 4 folate, to reduced enzyme first leaves the UV–vis spectrum relatively unchanged, however addition of the second substrate significantly changes the absorbance of the FDTS complex (black trace in Fig.…”