The effect of mutation on RNA Diels-Alderases

Chemical mutagenesis of a previously reported RNA Diels-Alderase (DA22) was followed by in vitro selection based on [4 + 2] catalysis. New mutated families of RNA Diels-Alderases closely related in sequence space were obtained. The mutated Diels-Alderases selected showed significant improvements in catalytic efficiency (k_cat/K_m) as compared to the original DA22. The improvement in catalytic activity was primarily due to a decrease in K _m, but modest increases in k_cat were also observed. The increase in catalytic activity of these new Diels-Alderases was found not to negatively affect their dienophile specificity. Surprisingly, one of the most active Diels-Alderases (DAM 40), a subtle sequence mutant of DA22, was found to show a new metal dependence and could function with Ni²⁺ as the only transition-metal ion. Truncation experiments of DA22 showed that the region shown to be hypervariable at the 3′-end of the structure could be deleted without a significant decrease in the relative rate of Diels-Alder catalysis.