**3.4 KARI structure analysis**

The KARI crystal structure is an asymmetric dimer. This latter is formed by one protomer in the holo-form due to the cofactors (Mg2+ and NADP(H)) binding, while the other is in the apo-form. As described previously for similar KARI enzymes, the dimerization is crucial for the construction of the active site, which is formed by some regions of the N-terminal domain and C-terminal domain of one subunit and the C-domain of the other subunit in the dimer. This arrangement is shared by other KARIs from other bacteria.

The KARI enzyme is composed of two distinct domains, the N-terminal (1–181) and C-terminal (182–327). The N-terminal domain, composed of alpha helix and beta sheets, harbor the NADPH-binding domain ilvN (14–177), with the binding sites for NADP (R48 and S52), while the catalytic domain ilvC is present in the C-terminal domain (183–326). The metal binding sites are present in both N-terminal and C-terminal domains (N39, V70, K71, and A73), and (D190 and E194), respectively. The structure of KARI binds four Mg2+ ions in the active sites and five Mg2+ on the surface of the protein.

A high electron density is shown in two locations deep inside the active site of KARI. This density is ascribed to magnesium ions coordinated differently to the structure. The first Mg2+ ion (Mg2+ (I)) is coordinated by the side chains of two residues (D188 and E192), and four water molecules, while the second Mg2+(II) is coordinated by the side chains of D188, E224 and E228, in addition to three water molecules. The metal ligands adopt an octahedron coordination geometry, with distances varying between 2.0 and 2.1 A between the metal and its ligand. The average B-factors for Mg2+(I) and Mg2+(II) are 11.5 Å2 and 10 Å2, respectively, supporting a highly ordered structure in this region of KARI [26].

Because of the absence of crystal structures of KARI from *N. gonorrhoeae*, the structure analysis is based on similar structure for close organisms having similar amino acid sequences. Based on the crystal structure of Mg KARI, the active site pocket and thus the magnesium ions are exposed to the solvent, allowing ready access to the substrates, the NADPH, or an inhibitor able to prevent their binding to the structure. The expected binding site for NADPH includes residues from Y22 to G26. All these residues are solvent accessible, hence the residue S24 seems to be the main entrance to the active site. Thus, the designed inhibitors should be designed that target this surface with metal coordination.
