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 Ni(2+) 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.