Abstract
Flavin-dependent thymidylate synthase (FDTS) ThyX that catalyzes the biosynthesis of TMP by the methylation of uracil moiety of dUMP is crucial for the growth of several important human pathogens including Helicobacter pylori, Mycobacteria tuberculosis, and Campylobacter jejuni. Given its uniqueness, ThyX has been recognized as a promising target for the development of anti-bacterial drug. Here, we determined the Campylobacter jejuni ThyX (CjThyX) structure to 3.0 Å resolution using single-wavelength anomalous dispersion methods on a mutant of CjThyX (A17MA49ML110M). Based on the mutant structure, we are able to solve the wild-type CjThyX．dUMP．FAD as well as A17MA49ML110M．dUMP．FAD to 2.2 Å and 2.45 Å resolution, respectively. The monomeric wild-type CjThyX structure is composed of 11 α-helices and 4 anti-parallel β-strands. Homotetramer is observed in which four FAD and dUMP molecules are situated at the interface of inter-subunits. In the active site, Ser84, Thr85, and Arg86 clamp the dUMP at a favor position through a helical shift. FAD is bound by the unique ThyX motif near the uridyl ring of dUMP. Structural comparisons with HpThyX and MtThyX indicate that the catalytic sites of ThyX enzyme in different species are highly conserved. The biosynthetic mechanism of CjThyX might also similar to HpThyX which is driven by attacking the C6 position of dUMP via the nitrogen atom N5 of the non-enzymatic nucleophile FADH2.