Figure 4. Two-metal-ion mechanism.

(A) Schematic cartoon illustration of a two-metal-ion mechanism as exemplified by nucleic-acid polymerases (green arrows). The two Mg²⁺ ions (yellow) are highlighted as circles and interactions with surrounding ligands are depicted as dashed red lines. R₁ represents the nucleoside moiety of the nucleotide triphosphate (blue), R₂ symbolizes the growing 3′-end of a DNA or RNA chain (orange), and E corresponds to the protein environment. The interaction between the 3′-hydroxyl group of the terminal nucleotide and Mg-A lowers the pK_a-value of the ligand and allows for its deprotonation by a general base designated B. Both Mg²⁺ ions share a common α-phosphate oxygen ligand, an interaction also maintained during the pentacovalent transition state that results after the nucleophilic attack onto the α-phosphate accomplished by the deprotonated 3′-hydroxyl group. As indicated by the purple dashed line, a basic amino-acid residue might act as general acid and protonate the additional negative charge at the β-phosphate group. (B) Schematic drawing of the proposed two-metal-ion mechanism in CMP-Kdo synthetases. R₃ represents the cytidine moiety of the CTP and R₄ symbolizes the C₈H₁₃O₇ portion of the Kdo sugar. The symbol and color scheme is the same as in (A). Amino-acid residues are labeled according to AA-LCKS nomenclature. Consistent with (A), the C2 hydroxyl group of the Kdo might coordinate to Mg-A, which could facilitate its subsequent deprotonation, and K19 might play the role of a general acid. In contrast to the situation in nucleic acid polymerases and as evident from the AA-LCKS crystal structure, the two metal ions do not share a common oxygen ligand and Mg-B is not in contact with the protein environment.