Control of Stereoselectivity in an Enzymatic Reaction by Backdoor Access

Wombacher, Richard; Keiper, Sonja; Suhm, Sandra; Serganov, Alexander; Patel, Dinshaw J.; Jäschke, Andres

doi:10.1002/anie.200503280

Cited by 36 publications

(47 citation statements)

References 35 publications

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“…To enter the pocket in this orientation, it must have squeezed through an opening that is, according to the X-ray structure, far too narrow (9). (2) It was recently demonstrated that, under certain conditions, the anthracene diene can enter the pocket from the ‘wrong’ side, through a small ‘back door’ opening, giving rise to an alternative stereochemical course of the reaction (44). Again, the dimensions of this orifice are too small for the substrate to gain access.…”

Section: Data Analysis and Discussionmentioning

confidence: 99%

Mg2+-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis

Kobitski¹,

Nierth²,

Helm³

et al. 2007

Nucleic Acids Research

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123

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Here, we report a single-molecule fluorescence resonance energy transfer (FRET) study of a Diels-Alderase (DAse) ribozyme, a 49-mer RNA with true catalytic properties. The DAse ribozyme was labeled with Cy3 and Cy5 as a FRET pair of dyes to observe intramolecular folding, which is a prerequisite for its recognition and turnover of two organic substrate molecules. FRET efficiency histograms and kinetic data were taken on a large number of surface-immobilized ribozyme molecules as a function of the Mg2+ concentration in the buffer solution. From these data, three separate states of the DAse ribozyme can be distinguished, the unfolded (U), intermediate (I) and folded (F) states. A thermodynamic model was developed to quantitatively analyze the dependence of these states on the Mg2+ concentration. The FRET data also provide information on structural properties. The I state shows a strongly cooperative compaction with increasing Mg2+ concentration that arises from association with several Mg2+ ions. This transition is followed by a second Mg2+-dependent cooperative transition to the F state. The observation of conformational heterogeneity and continuous fluctuations between the I and F states on the ∼100 ms timescale offers insight into the folding dynamics of this ribozyme.

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Section: Data Analysis and Discussionmentioning

confidence: 99%

Mg2+-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis

Kobitski¹,

Nierth²,

Helm³

et al. 2007

Nucleic Acids Research

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123

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“…Jäschke and coworkers have extensively studied a ribozyme that was identified for the Diels-Alder reaction between anthracene and maleimide substrates. [25,57] Primarily for comparison purposes, identification of deoxyribozymes for the Diels-Alder reaction was undertaken by Chandra and Silverman. [27] The RNA sequence of Jäschke's 39M49 ribozyme was partially randomized and prepared as DNA.…”

Section: Dna As a Catalystmentioning

confidence: 99%

“…[27] The sense of enantioselectivity is not known, although there is an appreciable degree of enantioselectivity (M. Chandra and S.K.S., unpublished data), as is the case for Jäschke's Diels-Alder ribozyme. [25,57] …”

Section: Figurementioning

confidence: 99%

DNA as a Versatile Chemical Component for Catalysis, Encoding, and Stereocontrol

Silverman

2010

Angew Chem Int Ed

231

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DNA (deoxyribonucleic acid) is the genetic material common to all of Earth's organisms. Our biological understanding of DNA is extensive and well-exploited. In recent years, chemists have begun to develop DNA for nonbiological applications in catalysis, encoding, and stereochemical control. This Review summarizes key advances in these three exciting research areas, each of which takes advantage of a different subset of DNA's useful chemical properties.

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“…The ( R , R )‐enantiomer of the product has been concluded to be more favorable by 16% of enantiomeric excess based on chemical studies. [15, 16] [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]…”

Section: Introductionmentioning

confidence: 99%

“…It has been recently found that the Diels‐Alderase ribozyme can be used to selectively synthesize both product enantiomers in one catalytic pocket by controlling access to the active site from opposite directions through different “doors”. [16] Indeed, the X‐ray crystal structure of the ribozyme:product complex revealed that the catalytic center is accessible from both “front” and “back” doors, involving openings of different sizes. [19] For the purpose of crystallization, the reaction was performed with the diene substrate covalently linked to the ribozyme by a short flexible tether, yielding a reaction product bound as the ( S , S )‐enantiomer (over 90% of enantiomeric excess), whereas the untethered catalytic reaction produces predominately the ( R , R )‐enantiomer (16% of enantiomeric excess),[15, 16] see Figure 1.…”

Section: Introductionmentioning

confidence: 99%

Stereoselection in the diels–alderase ribozyme: A molecular dynamics study

et al. 2012

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The Diels-Alderase ribozyme is an in vitro-evolved ribonucleic acid enzyme that catalyzes a [4 + 2] cycloaddition reaction between an anthracene diene and a maleimide dienophile. The ribozyme can in principle be used to selectively synthesize only one product enantiomer, depending on which of the two entrances to the catalytic pocket, "front" or "back", the substrate is permitted to use. Here, we investigate stereoselection and substrate recognition in the ribozyme by means of multiple molecular dynamics simulations, performed on each of the two substrates individually in the pocket, on the reactant state, and on the product state. The results are consistent with a binding mechanism in which the maleimide likely binds first followed by the anthracene, which enters preferentially through the front door. The free energy profiles for anthracene binding indicate that the pre-(R,R)-enantiomer conformation is slightly preferred, in agreement with the experimentally observed small enantiomeric excess of the (R,R)-enantiomer of the product. The reactant state is stabilized by the simultaneous presence of both substrates bound to their binding sites in the hydrophobic pocket as well as by stacking interactions between them.

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Control of Stereoselectivity in an Enzymatic Reaction by Backdoor Access

Cited by 36 publications

References 35 publications

Mg2+-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis

Mg2+-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis

DNA as a Versatile Chemical Component for Catalysis, Encoding, and Stereocontrol

Stereoselection in the diels–alderase ribozyme: A molecular dynamics study

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