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 (kcat/Km) as compared to the original DA22. The improvement in catalytic activity was primarily due to a decrease in Km, but modest increases in kcat 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 cata...