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Phosphatase-inert glucosamine 6-phosphate mimics serve as actuators of the glmS riboswitch.

Fei X, Holmes T, Diddle J, Hintz L, Delaney D, Stock A, Renner D, McDevitt M, Berkowitz DB, Soukup JK - ACS Chem. Biol. (2014)

Bottom Line: Interestingly, the malonyl ether (13, extra O atom) is much more effective than the simple malonate (17), and the "sterically true" phosphonate (5) is far superior to the chain-truncated (7) or chain-extended (11) analogs, suggesting that positioning via Mg coordination is important for activity.Docking results are consistent with this view.Indeed, the viability of the phosphonate and 6-O-malonyl ether mimics of GlcN6P points to a potential new strategy for artificial actuation of the glmS riboswitch in a biological setting, wherein phosphatase-resistance is paramount.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Nebraska , Lincoln, Nebraska 68588, United States.

ABSTRACT
The glmS riboswitch is unique among gene-regulating riboswitches and catalytic RNAs. This is because its own metabolite, glucosamine-6-phosphate (GlcN6P), binds to the riboswitch and catalytically participates in the RNA self-cleavage reaction, thereby providing a novel negative feedback mechanism. Given that a number of pathogens harbor the glmS riboswitch, artificial actuators of this potential RNA target are of great interest. Structural/kinetic studies point to the 2-amino and 6-phosphate ester functionalities in GlcN6P as being crucial for this actuation. As a first step toward developing artificial actuators, we have synthesized a series of nine GlcN6P analogs bearing phosphatase-inert surrogates in place of the natural phosphate ester. Self-cleavage assays with the Bacillus cereus glmS riboswitch give a broad SAR. Two analogs display significant activity, namely, the 6-deoxy-6-phosphonomethyl analog (5) and the 6-O-malonyl ether (13). Kinetic profiles show a 22-fold and a 27-fold higher catalytic efficiency, respectively, for these analogs vs glucosamine (GlcN). Given their nonhydrolyzable phosphate surrogate functionalities, these analogs are arguably the most robust artificial glmS riboswitch actuators yet reported. Interestingly, the malonyl ether (13, extra O atom) is much more effective than the simple malonate (17), and the "sterically true" phosphonate (5) is far superior to the chain-truncated (7) or chain-extended (11) analogs, suggesting that positioning via Mg coordination is important for activity. Docking results are consistent with this view. Indeed, the viability of the phosphonate and 6-O-malonyl ether mimics of GlcN6P points to a potential new strategy for artificial actuation of the glmS riboswitch in a biological setting, wherein phosphatase-resistance is paramount.

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GlcN6P analog-actuated glmS ribozyme self-cleavage.Shown are products of reactions incubated for varying times in thepresence of 10 mM analog. Bands correspond to the ribozyme (open arrowhead)and its 3′-cleavage product (filled arrowhead). Note: Datafor the less active analogs is in the SI.
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fig8: GlcN6P analog-actuated glmS ribozyme self-cleavage.Shown are products of reactions incubated for varying times in thepresence of 10 mM analog. Bands correspond to the ribozyme (open arrowhead)and its 3′-cleavage product (filled arrowhead). Note: Datafor the less active analogs is in the SI.

Mentions: The BacilluscereusglmS ribozyme was utilizedin self-cleavage kinetic assays performedin the presence of various synthesized GlcN6P analogs. Nine analogswere tested for their ability to support glmS self-cleavage(Figure 8). Seven of the nine analogs containan unmodified amine functionality. The remaining two analogs combinethe “sterically true” phosphonate ester with N-methylatedamino groups. Mono- or dimethylation of the amine still retains thelone pair on nitrogen, which would, in principle, still allow theamine to function as a general base/acid.


Phosphatase-inert glucosamine 6-phosphate mimics serve as actuators of the glmS riboswitch.

Fei X, Holmes T, Diddle J, Hintz L, Delaney D, Stock A, Renner D, McDevitt M, Berkowitz DB, Soukup JK - ACS Chem. Biol. (2014)

GlcN6P analog-actuated glmS ribozyme self-cleavage.Shown are products of reactions incubated for varying times in thepresence of 10 mM analog. Bands correspond to the ribozyme (open arrowhead)and its 3′-cleavage product (filled arrowhead). Note: Datafor the less active analogs is in the SI.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4273988&req=5

fig8: GlcN6P analog-actuated glmS ribozyme self-cleavage.Shown are products of reactions incubated for varying times in thepresence of 10 mM analog. Bands correspond to the ribozyme (open arrowhead)and its 3′-cleavage product (filled arrowhead). Note: Datafor the less active analogs is in the SI.
Mentions: The BacilluscereusglmS ribozyme was utilizedin self-cleavage kinetic assays performedin the presence of various synthesized GlcN6P analogs. Nine analogswere tested for their ability to support glmS self-cleavage(Figure 8). Seven of the nine analogs containan unmodified amine functionality. The remaining two analogs combinethe “sterically true” phosphonate ester with N-methylatedamino groups. Mono- or dimethylation of the amine still retains thelone pair on nitrogen, which would, in principle, still allow theamine to function as a general base/acid.

Bottom Line: Interestingly, the malonyl ether (13, extra O atom) is much more effective than the simple malonate (17), and the "sterically true" phosphonate (5) is far superior to the chain-truncated (7) or chain-extended (11) analogs, suggesting that positioning via Mg coordination is important for activity.Docking results are consistent with this view.Indeed, the viability of the phosphonate and 6-O-malonyl ether mimics of GlcN6P points to a potential new strategy for artificial actuation of the glmS riboswitch in a biological setting, wherein phosphatase-resistance is paramount.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Nebraska , Lincoln, Nebraska 68588, United States.

ABSTRACT
The glmS riboswitch is unique among gene-regulating riboswitches and catalytic RNAs. This is because its own metabolite, glucosamine-6-phosphate (GlcN6P), binds to the riboswitch and catalytically participates in the RNA self-cleavage reaction, thereby providing a novel negative feedback mechanism. Given that a number of pathogens harbor the glmS riboswitch, artificial actuators of this potential RNA target are of great interest. Structural/kinetic studies point to the 2-amino and 6-phosphate ester functionalities in GlcN6P as being crucial for this actuation. As a first step toward developing artificial actuators, we have synthesized a series of nine GlcN6P analogs bearing phosphatase-inert surrogates in place of the natural phosphate ester. Self-cleavage assays with the Bacillus cereus glmS riboswitch give a broad SAR. Two analogs display significant activity, namely, the 6-deoxy-6-phosphonomethyl analog (5) and the 6-O-malonyl ether (13). Kinetic profiles show a 22-fold and a 27-fold higher catalytic efficiency, respectively, for these analogs vs glucosamine (GlcN). Given their nonhydrolyzable phosphate surrogate functionalities, these analogs are arguably the most robust artificial glmS riboswitch actuators yet reported. Interestingly, the malonyl ether (13, extra O atom) is much more effective than the simple malonate (17), and the "sterically true" phosphonate (5) is far superior to the chain-truncated (7) or chain-extended (11) analogs, suggesting that positioning via Mg coordination is important for activity. Docking results are consistent with this view. Indeed, the viability of the phosphonate and 6-O-malonyl ether mimics of GlcN6P points to a potential new strategy for artificial actuation of the glmS riboswitch in a biological setting, wherein phosphatase-resistance is paramount.

Show MeSH