Detection of Intermediates in the Oxidative Half-Reaction of the FAD-dependent Thymidylate Synthase from Thermotoga maritima: Carbon Transfer without Covalent Pyrimidine Activation.

Biochemistry

PubMedID: 25068636

Conrad JA, Ortiz-Maldonado M, Hoppe SW, Palfey BA. Detection of Intermediates in the Oxidative Half-Reaction of the FAD-dependent Thymidylate Synthase from Thermotoga maritima: Carbon Transfer without Covalent Pyrimidine Activation. Biochemistry. 2014;.
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 an FAD 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 analogs 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 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.