Functional evidence for active site location of tetrameric thymidylate synthase X at the interphase of three monomers

Abstract

Little is known about the catalytic mechanism of the recently discovered ThyX family of flavin-dependent thymidylate synthases that are required for thymidylate (deoxythymidine 5'-monophosphate) synthesis in a large number of microbial species. Using a combination of site-directed mutagenesis and biochemical measurements, we have identified several residues of the Helicobacter pylori ThyX protein with crucial roles in ThyX catalysis. By providing functional evidence that the active site(s) of homotetrameric ThyX proteins is formed by three different subunits, our findings suggest that ThyX proteins have evolved through multimerization of inactive monomers. Moreover, because the active-site configurations of ThyX proteins, present in many human pathogenic bacteria, and of human thymidylate synthase ThyA are different, our results will aid in the identification of compounds specifically inhibiting microbial growth.

Fig. 1.

Sequence alignment of a diverse set of ThyX homologs is shown. The alignment was obtained with the use of clustalx program using default settings (19) followed by manual modifications. Conserved residues within 7 Å of the redox-active N5 atom of an FAD molecule are indicated by the asterisk. The secondary structure elements (cylinders, α-helices; arrows, β-strands) of T. maritima ThyX proteins are indicated above the alignment (4). The residues investigated in this study are indicated by the red background. The green dotted line indicates a central variable region of ThyX proteins that participates in the stabilization of ThyX tertiary structure (see also Fig. 5B).

Fig. 5.

(A) ThyX tetramer (4) where the different subunits are shown with the different colors. FAD, S88, H53, and R78 (corresponding to active-site residues of H. pylori ThyX identified in this work) are represented with balls and sticks. (B) The central variable part of the ThyX protein indicated with the green dotted line in Fig. 1 is indicated by the different colors by monomer. (C) Only the conserved residues located within 7 Å of the redox-active N5 atom of the FAD molecule (only one of four FAD molecules of homotetramer is shown) are represented (see also Fig. 1). (D) The active-site configuration of T. maritima ThyX protein. A FAD molecule, Arg-78, Ser-88, and His-53 (T. maritima ThyX numbering) corresponding, respectively, to Ser-84, His-48, and Arg-74 in H. pylori protein are represented by balls and sticks. The different subunits of ThyX homotetramer are shown with the different colors. Note that the residues from the three different subunits participate in the formation of the single active site.

Fig. 2.

The ability of wild-type or mutated H. pylori thyX genes to permit thymidine-independent growth of E. coli thymidine auxotroph χ2913 (ΔthyA) was scored after 3 days on M9 minimal agar lacking thymidine in the presence and absence of 0.2% l-arabinose.

Fig. 3.

(A) SDS/10% PAGE of H. pylori wild-type (wt) and mutated ThyX proteins by using ≈1 μg of purified protein stained with Coomassie Brilliant Blue. M1 and M24 refer to the alternative start codons of H. pylori HP1533. (B) Spectroscopic analysis of H. pylori ThyX wild-type and mutant proteins (1). (C) “Deprotonation” activity of H. pylori ThyX proteins with 6.25 μM dUMP at 37°C as described in Materials and Methods. Complete reaction mixtures contained 50 mM Hepes (pH 7.0), 10% glycerol, 0.9 mM CH₂H₄folate, 10 mM MgCl₂, 2 mM NADPH, 1 mM NADH, 0.5 mM FAD, and 6.25 μM [5-³H]dUMP (specific activity, 2.55 Ci/mmol). The specific activity for the wild-type protein corresponding to 0.95 nmol of ³H₂O formed per min per mg of protein was arbitrarily chosen to correspond to 100%.

Fig. 4.

Maximum specific activities for ThyX (dUMP) wild-type (wt) and mutants were measured under single-turnover conditions. The reaction conditions are as described in Fig. 3C, except various dUMP concentrations were used.