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 ± 1.25 μM and 0.35 ± 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.

Figure 1

Purity (A) and mass spectral identification (B) of the recombinant HpThyX. Purified HpThyX ran as a single band on 10% SDS-PAGE (A). Left lane, purified ThyX protein. Right lane, molecular weight standard marker. Molecular mass in kDa is indicated on the right.

Figure 2

Enzymatic kinetics characterization of recombinant HpThyX. Kinetic analyses were performed for (A) dUMP, (B) CH₂H₄folate, and (C) NADPH. Enzymatic activities of HpThyX were determined at different concentrations of the indicated substrates and cofactors, with all other substrates at fixed saturated concentrations as described in “Experimental Procedures” Section. (D) The enzymatic activities of recombinant HpThyX at different pH values. The assays were carried out in standard reaction mixture at a pH range from 6.5 to 10.5. The recombinant HpThyX showed its maximal activity at pH 8.5, and then, the activity of the enzyme at pH8.5 was taken as 1.0.

Scheme 1

The equation of ThyX catalyzed reaction. ThyX enzyme activity was measured by detecting the amount of ³H in the generated H₂O, the substrate dUMP was labeled with ³H, R = 2′-deoxyribose-5′-phosphate, R′ = p-aminobenzoyl-glutamate.

Figure 3

Overall structure of HpThyX. (A) Tetramer structure. Subunits A, B, C, and D are colored green, cyan, yellow, and magenta, respectively. (B) Monomer structure. Superimposition of chains A, B, C, and D. The color scheme is the same as that of Figure 2A.

Figure 4

Active site structure of HpThyX. The cofactor-binding site of HpThyX composed of subunits A, B, and D, which colored green, cyan, and magenta, respectively, and hydrogen bonds are presented as dotted lines (same below). The substrate-binding site of HpThyX formed by residues from subunits A and B, the color scheme is the same as that of Figure 3A.

Figure 5

Key active sites in HpThyX reaction. The side-chains of Ser84 and Ser85 are 4.13 and 7.04 Å away from C6 of dUMP, respectively. N5 of FAD is only 3.37 Å away from C6 of dUMP. Hydrogen bonds are presented as dotted lines in red.

Figure 6

Structure-based sequence alignment of HpThyX, TmThyX, and MtbThyX. PDB codes are indicated beside the protein names. Secondary structures of HpThyX are labeled. Residues involved in the binding network are labeled, and the alignment was produced by the program ESPript.

Figure 7

Structures of flavoenzyme-FAD-substrate complex. HpThyX(PDB:3N3Y), TmThyX(PDB:1O26), MtbThyX(PDB:2AF6), and MtbUGM(PDB:3INT). The distances between N5 of FAD and C6/C1 of substrate are 3.37Å, 3.48 Å, 3.35 Å, and 3.57 Å for HpThyX, TmThyX, MtbThyX, and MtbUGM, respectively, whereas the corresponding angles C5/C2(substrate)–C6/C1(substrate)–N5(FAD) are 75.4°, 78.0°, 69.7° and 74.0°.