RESUMO
Ribox02 is an in-vitro evolved RNA molecule that exhibits the activity analogous to that of alcohol dehydrogenase, catalyzing redox reactions in a cofactor dependent manner. It forms a compact pseudo-knotted structure and oxidizes an alcohol seven orders of magnitude faster than the estimated spontaneous rate. To gain insights into the structure-function relationship of Ribox02, we firstly performed mutational studies to identify the critical nucleotide bases for catalysis and then engineered the ribozyme structure to improve its overall fold. The re-engineered Ribox02, named Robust, was able to catalyze the reaction approximately 25% more efficiently, leading us to generate a transacting system.
Assuntos
Álcool Desidrogenase/química , RNA Catalítico/química , Álcool Desidrogenase/metabolismo , Sequência de Bases , Sítios de Ligação , Evolução Molecular Direcionada , Dados de Sequência Molecular , Oxirredução , RNA Catalítico/metabolismo , Relação Estrutura-AtividadeRESUMO
We report here the ability of an alcohol dehydrogenase (ADH) ribozyme to reduce a benzaldehyde. While the ribozyme was initially evolved in vitro based on the activity for the NAD+-dependent oxidation of the benzyl alcohol, we found that this ADH ribozyme is also capable of reducing the aldehyde in the presence of NADH and Zn2+. The rate acceleration gained by ribozyme catalysis was more than 6 orders of magnitude larger than the spontaneous reaction. Although the reversibility of phosphordiester and acyl transfer reactions catalyzed by ribozymes was known, that of other chemical reactions has not been well established. This study has demonstrated the reversibility of a hydride transfer chemistry catalyzed by the ADH ribozyme. Most interestingly, the ribozyme shares many features with the protein ADHs, e.g., reversibility and NADH/Zn2+ dependence.
Assuntos
Álcool Desidrogenase/metabolismo , Aldeídos/metabolismo , NAD/química , RNA Catalítico/metabolismo , Zinco/química , Álcool Desidrogenase/química , Álcool Desidrogenase/genética , Aldeídos/química , Sequência de Bases , Benzaldeídos/química , Álcool Benzílico/química , Catálise , Endorribonucleases , Cinética , Dados de Sequência Molecular , Oxirredução , Radioisótopos de Fósforo , RNA Catalítico/químicaRESUMO
We report an RNA molecule that exhibits activity analogous to that of alcohol dehydrogenase (ADH). Directed in vitro evolution was used to enrich nicotinamide adenine dinucleotide (NAD+)-dependent redox-active RNAs from a combinatorial pool. The most active ribozyme in the population forms a compact pseudoknotted structure and oxidizes an alcohol seven orders of magnitude faster than the estimated spontaneous rate. Moreover, this ADH RNA was coupled with a redox relay between NADH and flavin adenine dinucleotide to give a NAD+-regeneration system. Our demonstration of the redox ability of RNA adds support to an RNA-based metabolic system in ancient life.
