Crystal structure and enzymatic characterization of thymidylate synthase X from Helicobacter pylori strain SS1

Abstract

Thymidylate synthase X (ThyX) catalyzes the methylation of dUMP to form dTMP in bacterial life cycle and is regarded as a promising target for antibiotics discovery. Helicobacter pylori is a human pathogen associated with a number of human diseases. Here, we cloned and purified the ThyX enzyme from H. pylori SS1 strain (HpThyX). The recombinant HpThyX was discovered to exhibit the maximum activity at pH 8.5, and K m values of the two substrates dUMP and CH 2 H 4 folate were determined to be 15.3 6 1.25 lM and 0.35 6 0.18 mM, respectively. The analyzed crystal structure of HpThyX with the cofactor FAD and the substrate dUMP (at 2.31 Å ) revealed that the enzyme was a tetramer bound to four dUMP and four FAD molecules. Different from the catalytic feature of the classical thymidylate synthase (ThyA), N5 atom of the FAD functioned as a nucleophile in the catalytic reaction instead of Ser84 and Ser85 residues. Our current work is expected to help better understand the structural and enzymatic features of HpThyX thus further providing valuable information for anti-H. pylori inhibitor discovery.

Folate binding site of flavin-dependent thymidylate synthase

“…Nevertheless, the flavin occupying this unique conformation showed better electron density than the other isoalloxazine moieties of FAD in the tetramer. As it has been pointed out earlier, the isoalloxazine of FAD is either disordered or shows very weak electron density in structures without dUMP (7,9,15,21,30). Interestingly, the newly observed binding site of the isoalloxazine moiety is almost identical to the binding site of the pterin moiety of the folate derivatives presented in Fig.…”

“…1). Although several studies have published structures of FDTS from various organisms in complex with dUMP and dUMP analogs along with FAD (7,9,15,21,30), and even a nonreactive complex with NADP + (29), a structure with CH 2 H 4 folate or any other folates has been an unrealized goal. Here we report the structures of the TmFDTS with the cofactor CH 2 H 4 folate and other folate derivatives in complex with FAD and dUMP (Fig.…”

“…This observation led to the identification of alternative flavin-dependent thymidylate synthases (FDTSs), which are encoded by the thyX gene and have no sequence or structure homology to classic TSase enzymes (2,6,7). Furthermore, multiple studies have identified key differences in the molecular mechanism of catalysis between FDTSs and classic TSases (2,4,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). These differences, along with the fact that the thyX gene is present in many human pathogens (e.g., bacteria causing Anthrax, Tuberculosis, Typhus, and other diseases), renders these flavo-enzymes as potential targets for rational inhibitor design, possibly affording compounds that might be effective antimicrobial drugs (11,(22)(23)(24).…”

“…These studies may have been complicated by the competing oxidase activity (FADH 2 reaction with O 2 to form H 2 O 2 ) (10,14,27) and by other kinetic complexities, such as possible positive cooperativity (15). Published structures of FDTSs (7,9,15,29,30) have not been reported with folates, making further differentiation between these methylene transfer mechanisms difficult. Structural information regarding folate binding would not only provide a better understanding of the methylene transfer step, but will also present a new FDTS binding mode that might serve as a potential target for inhibitor design.…”

Mechanisms and inhibition of uracil methylating enzymes

“…1B). Since then, crystal structures for FDTSs from Mycobacterium tuberculosis , 30 Paramecium bursaria chlorella virus-1, 31 and more recently Helicobacter pylori 32 have been solved, with FAD only and FAD in combination with dUMP, 5F-dUMP or 5Br-dUMP. Additionally, a structure with NADP + has been solved for M. tuberculosis FDTS where nicotinamide replaced the flavin cofactor in the enzyme during crystallization.…”