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 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.
Tarasow
Theodore M
TM
Department of Chemistry, College of Physical and Mathematical Sciences, North Carolina State University, Raleigh, North Carolina 27695-8204, USA.
Kellogg
Elizabeth
E
Holley
Benjamin L
BL
Nieuwlandt
Daniel
D
Tarasow
Sandra L
SL
Eaton
Bruce E
BE
D001483:Base Sequence; D002384:Catalysis; D002412:Cations; D017124:Conserved Sequence; D015723:Gene Library; D007700:Kinetics; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D002412:Cations; D008670:Metals; D016337:RNA, Catalytic