Research Spotlight: Insights into glmS ribozyme catalysis

By Matt Wargo • June 6th, 2011

Citation: Brooks KM and Hampel KJ. 2011. Rapid steps in the glmS ribozyme catalytic pathway: cation and ligand requirements. Biochemistry 50(13):2424-2433

Direct Link to Publication

Authors’ Association with MMG:
Krista Brooks – is a recent MMG graduate student now a postdoc at Scripps
Ken Hampel – is an Assistant Professor in the MMG Department

Summary:
Riboswitches are regions of bacterial and eukaryotic mRNAs that function as metabolite-sensing genetic switches. The glmS ribozyme is a broadly distributed Gram-positive bacterial riboswitch that senses the intercellular concentration of glucosamine 6-phosphate. Upon binding this metabolite the glmS ribozyme catalyzes a site-specific RNA cleavage within the 5’UTR of the glmS mRNA. Cleavage of the 5’ UTR exposes a 5’ hydroxyl-terminated mRNA to the rapid degradation by 5’-3’ RNases. The result of the action of this RNA switch, therefore, is to mediate feedback repression of the GlmS protein, L-glutamine:D-fructose-6-phosphate aminotransferase. The ligand binding specificity is therefore central to the function of the riboswitch.

The requirements for rapid binding of the ligand were probed by kinetic measurements of the cleavage reaction under conditions where native folding of the RNA, which is slow and rate-limiting for the reaction (Brooks and Hampel, 2009), could be carried out as a preliminary step. Our data indicate that rapid ligand binding and catalysis requires divalent metal ions and that this requirement is linked to the presence of the phosphate moiety of the ligand. The absence of either Mg2+ or the ligand phosphate result in identical, non-additive losses in catalytic activity. In addition, we show that the glmS ribozyme is able to fold into its native conformation in the presence of high concentrations of monovalent cations. Our data therefore show no obligatory role for divalent cations outside of the ligand binding pocket.

Impact and Significance:
The GlmS enzyme catalyzes the rate-limiting step in the production of the UDP-N-acetylglucosamine (UDP-GlcNAc), a precursor of cell wall constiuents peptidoglycan and lipopolysaccharides, in Gram negative and positive bacteria. This pathway is the target of several established antibiotics and continues to be an attractive target for new antibiotic research. Several important Gram positive bacterial human pathogens utilize the glmS riboswitch to control this pathway, including S. aureus, Listeria momocytogenese, and B. anthracis. Our data may suggest new approaches to the development of antibiotics which target this genetic switch.

Our data also impact our understanding of the biochemistry of bacterial riboswitches. The natural ligand of the glmS ribozyme, GlcN6P, is believed to be a coenzyme in the site-specific RNA cleavage reaction. Since RNA has a limited enzymatic repertoire it has been speculated that one way in which RNAs could have carried out a broad range of metabolic chemistry in a hypothesized “RNA world” is to utilize coenzymes. Indeed, many bacterial riboswitches bind protein coenzymes such as S-adenosyl methionine, flavin mononucleotide and cobalamin. However, only the glmS ribozyme has been shown to use a coenzyme in the mechanistic reaction chemistry. Our data demonstrate essential coenzyme binding requirements for the glmS ribozyme.

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