The hepatitis delta virus ribozyme catalyzes an RNA cleavage reaction involving a catalytic nucleobase and a divalent metal ion. The catalytic base, C75, serves as a general acid and has a pKa shifted towards neutrality. A recent crystal structure of the pre-cleavage ribozyme identified a Mg2+ ion that interacts through its partial hydration sphere with the G25�U20 reverse wobble.  In addition, this Mg2+ ion is in position to directly coordinate the nucleophile, the 2'-hydroxyl of U(-1), suggesting it can serve as a Lewis acid to facilitate deprotonation of the 2'-hydroxylj. Although the active site Mg2+ion appears to be poised to participate in the cleavage reaction, there is no experimental evidence that demonstrates such a role for the Mg2+ ion. To test the role of the active site Mg2+ ion, we replaced the G25�U20 reverse wobble with an isosteric A25�C20 reverse wobble.  Solution biochemical characterization, molecular dynamics simulations, and electrostatic calculations all suggest that the A25�C20 double mutant HDV ribozyme folds correctly but is unable to position the active site Mg2+ ion such that it can participate in the cleavage reaction. In the crystal structure, the active site Mg2+ ion also interacts with the pro-RP oxygen of the scissile phosphate. Replacing the pro-RP oxygen of the scissile phosphate with a sulfur atom is detrimental to catalysis, however this substitution can be rescued in the presence of a thiophilic metal ion. These approaches link the active site Mg2+ ion observed in the crystal structure to a catalytic role in HDV ribozyme catalysis.

Identification of the catalytic Mg<sup>2+</sup> ion in the HDV ribozyme

245th National Meeting (2013)

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Identification of the catalytic Mg2+ ion in the HDV ribozyme

Identification of the catalytic Mg²⁺ ion in the HDV ribozyme