Diels-Alderase ribozymes

Stuhlmann, F., Jaschke, A. Chai acterization of an RNA active site interactions between a Diels-Alderase ribozyme and its substrates and products. J. Am. Chem. Soc. 124, 3238-3244 (2002). 

The secondary double-reciprocal plot of the data from Figure 5.3.5 gives Mi-chaelis constants of 0.37 mM for the diene and 8 nm for the dienophile. The calculated maximum rate Vmax is 0.15 mM min-1, which at a ribozyme concentration of 7 xm corresponds to a kcat of 21 min-1. The highest initial rate that was measured directly corresponds to a catalytic turnover of approximately 6 min-1. With these catalytic properties the 49nt Diels-Alderase ribozyme is among the faster... 

An RNA Diels-Alderase ribozyme recently developed that catalyses the formation of carbon-carbon bonds between a tethered anthracene diene and a biotinylated maleimide dienophile. The ribozyme active site has been further characterised by chemical substitution of the diene and dienophile. It was shown that the diene must be an anthracene, and substitution only at specific sites is permitted. The dienophile must be a maleimide with an unsubstituted double bond. The RNA-diene interaction was found to be governed preferentially by stacking interactions. A ribozyme has been selected that catalyses the synthesis of dipeptides using an aminoacyl-adenylate substrate. The ribozyme catalysed the formation of 30 different dipeptides, many with rates similar to that of the Met-Phe dipeptide used in the selection process. 

Similar CW EPR titration experiments have been performed by Schiemann and coworkers on the Diels-Alderase ribozyme [89]. It was demonstrated that five high-affinity Mn binding sites with an upper Kj of 0.6 0.2 pM exist in this ribozyme. From the spectral changes on stoichiometric mixtures of Mn and Cd the binding sites could be assigned to three different types inner sphere, outer sphere, and a dimeric site. 

Catalysis of C—C Bond Formation by Diels-Alderase Ribozymes... 

Another characteristic feature of enzymatic catalysis was demonstrated for the Diels-Alderase ribozymes, namely enantioselective bond formation. While the uncatalysed... 

Figure 14.5 Crystal structure of the Diels-Alderase ribozyme. a) Ribozyme secondary structure with helices I, II and III, the asymmetric bubble, and the conserved 5 end.

The structure of the free Diels-Alderase ribozyme was found to be virtually identical to the structure of the product complex, providing strong support for the concept of a preformed catalytic pocket, established by chemical probing. The anthracene-RNA conjugate could also be crystallized, however, the anthracene module and its linker were found to be disordered in the crystal. The RNA mapped well with the other crystal structures. 

Figure 14.7 Summarized structural requirements of dienes and dienophiles for acceptance by the Diels-Alderase ribozyme. Substitutions shown in black are tolerated, while those in outline letters are deleterious. A removal of whole substituent. ...

Figure 14.8 Proposed model for the catalytic mechanism of the Diels-Alderase ribozyme. a. Empty catalytic pocket, b. Michaelis complex with both substrates bound, c. Transition state inside the catalytic pocket, d. Ribozyme-bound product. Panels a and d are derived directly from the crystallographic data, panels b and c were obtained by manually docking the two substrates and the transition state, respectively, into the pocket.

S. Keiper, D. Bebenroth, B. Seelig, E. Westhof, A. Jaschke, Architecture of a Diels-Alderase ribozyme with a preformed catalytic pocket, Chem. Biol., 2004, 11, 1217—1227. 

A. Y. Kobitski, A. Nierth, M. Helm, A. Jaschke, G. U. Nienhaus, Mg -dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis. Nucleic Acids Res., 2007, 35, 2047-2059. 

Diels-Alderase ribozymes (DAR), isolated from a combinatorial RNA library, cause a (2 X 10 )-fold acceleration of the Diels-Alder cycloaddition of anthracene covalently tethered to ribozyme and a biotinylated maleimide in aqueous-buffered medium (Scheme 5.15). Jaschke recently reported the action of Diels-Alderase ribozymes as true catalysts, in the sense that they catalyze the cycloaddition of anthracene that is not covalently tethered to RNA and biotin maleimide in aqueous-buffered medium. 

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