title: Phosphatase-inert glucosamine 6-phosphate mimics serve as actuators of the glmS riboswitch.
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.
authors.affiliation: Department of Chemistry, University of Nebraska , Lincoln, Nebraska 68588, United States.
authors.forename: Xiang
authors.initials: X
authors.lastname: Fei
authors.forename: Thomas
authors.initials: T
authors.lastname: Holmes
authors.forename: Julianna
authors.initials: J
authors.lastname: Diddle
authors.forename: Lauren
authors.initials: L
authors.lastname: Hintz
authors.forename: Dan
authors.initials: D
authors.lastname: Delaney
authors.forename: Alex
authors.initials: A
authors.lastname: Stock
authors.forename: Danielle
authors.initials: D
authors.lastname: Renner
authors.forename: Molly
authors.initials: M
authors.lastname: McDevitt
authors.forename: David B
authors.initials: DB
authors.lastname: Berkowitz
authors.forename: Juliane K
authors.initials: JK
authors.lastname: Soukup
mesh_terms: D000900:Anti-Bacterial Agents / Q000138:chemical synthesis / Q000737:chemistry*; D001409:Bacillus cereus / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000819:agonists* / Q000737:chemistry; D001483:Base Sequence; D055162:Biocatalysis; D018360:Crystallography, X-Ray; D015964:Gene Expression Regulation, Bacterial; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D007700:Kinetics; D008314:Malonates / Q000138:chemical synthesis / Q000737:chemistry*; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D063065:Organophosphonates / Q000138:chemical synthesis / Q000737:chemistry*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D011485:Protein Binding; D059367:RNA Cleavage; D016337:RNA, Catalytic / Q000819:agonists* / Q000737:chemistry; D058928:Riboswitch / Q000235:genetics; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D005944:Glucosamine; D019298:Glucose-6-Phosphate; D008314:Malonates; D063065:Organophosphonates; D010744:Phosphoric Monoester Hydrolases; D016337:RNA, Catalytic; D058928:Riboswitch; C001293:glucosamine 6-phosphate; C000731191:component S, glutamate mutase protein, Bacteria; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)