Figure 5. Nucleotide binding site and the catalysis.
(A) dTMP binding pocket between the DHH domain and DHHA1 domain. dTMP is shown as stick (orange). Two conserved residues His397 and Arg393 at the entrance and exit to the active site are labeled and shown as sticks. (B) Close-up of the active site of drRecJ-dTMP (complex I). Two Mn2+ ions (A and B; magenta) are coordinated by a water molecule (red sphere) and conserved Asp and His residues (magenta dashed lines). Asp158 forms a hydrogen bond with His160, as indicated by the pink dashed line. The phosphate of the dTMP is held by Arg109, Arg280, Ser371 and Arg373 (blue dashed lines). (C) Catalytic efficiency of wild-type and mutant drRecJ as a bar graph showing the relative severity of the mutations. The Michaelis-Menten kinetics data are from Table 2. Data are represented as mean ± SEM. (D) Close-up of the active of site of complex III. The scissile phosphate centered on two catalytic Mn2+ is highlighted in cyan. The red arrowhead indicates the position of P-O bond breakage. Interaction between His397 and the oxygen atom of the scissile phosphate group is indicated by blue dashed line. (E) A comparison of the active sites in the complex I (white) with complex III (same color as in panel D). The nucleophilic water molecule observed in complex III occupies the position close to the His160 in drRecJ-dTMP structure (complex I). The red arrowhead indicates the position of P-O bond breakage and the black arrowhead indicates the direction of nucleophilic attack.
Figure 5—figure supplement 1. Denaturing PAGE gel showing the inactivation of mutant drRecJ proteins (alanine substitutions of residues involved in metal-ion-chelation).
