Figure 5. Overlay of the Irp3 and KijD10 active sites, highlighting differences in loops (A) and residues (B).

The colors are maintained in the (A) and (B) panels: NADP⁺-bound Irp3 is shown in green cartoon. KijD10 (PDB code 3RC1) is shown in pink cartoon. The NADP⁺ bound in the active sites are shown as yellow (Irp3) and magenta (KijD10) sticks. The substrate analogue, dTDP-benzene, for KijD10 is also shown in magenta sticks. KijD10 is a member of the glucose-fructose oxidoreductase family, and contains the GGX₃DX₃(Y/H) consensus and following loop shown as the grey cartoon, similar to Figure 4. The D-3 loop of Irp3 is shown in yellow. The J-K loop of Irp3 is shown in orange. The blue cartoon is for the M’-N’ loop of the opposing dimer, which would be connected by six disordered residues (256-261). (A) The grey loop in KijD10 undergoes a disorder to order transition when the protein goes from the open to the closed conformation, and with this transition, the nicotinamide of the NADP⁺ flips from syn to anti (33). The nicotinamide ring in Irp3 is anti as seen in the closed conformation of KijD10 shown here. The loop in Irp3 that undergoes a disorder-to-order transition upon NADP⁺-binding is the D-3 loop (yellow). The orange (J-K) loop of Irp3 is in a comparable position to the grey loop of KijD10, but does not contain the consensus sequence nor does it connect comparable secondary structure (this is the G-6 loop in Irp3) as discussed in Figure 4. We hypothesize that the very long M’-N’ loop (blue) resides in a different location in the presence of substrate and aids in binding the protein HMWP1 to which the substrate is covalently attached. (B) The EKP sequence of KijD10 and the corresponding EHP of Irp3 are shown in sticks and labeled. The K/H at the center of this loop is hypothesized to be the general acid, which is supported by mutagenic analyses in KijD10 (33). The Y186 of the GGX₃DX₃(Y/H) consensus sequence in KijD10 is displayed in sticks to show the relative location to S166, the structurally equivalent amino acid, and Y128, found in the linker between the N-terminal and C-terminal domains (the F-5 loop), both in Irp3. Labels in bold are for Irp3 and those in plain text are for KijD10.