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The GlcN6P cofactor plays multiple catalytic roles in the glmS ribozyme

Nature Chemical Biology volume 13, pages 439–445 (2017)Cite this article

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Abstract

RNA enzymes (ribozymes) have remarkably diverse biological roles despite having limited chemical diversity. Protein enzymes enhance their reactivity through recruitment of cofactors; likewise, the naturally occurring glmS ribozyme uses the glucosamine-6-phosphate (GlcN6P) organic cofactor for phosphodiester bond cleavage. Prior structural and biochemical studies have implicated GlcN6P as the general acid. Here we describe new catalytic roles of GlcN6P through experiments and calculations. Large stereospecific normal thio effects and a lack of metal-ion rescue in the holoribozyme indicate that nucleobases and the cofactor play direct chemical roles and align the active site for self-cleavage. Large stereospecific inverse thio effects in the aporibozyme suggest that the GlcN6P cofactor disrupts an inhibitory interaction of the nucleophile. Strong metal-ion rescue in the aporibozyme reveals that this cofactor also provides electrostatic stabilization. Ribozyme organic cofactors thus perform myriad catalytic roles, thereby allowing RNA to compensate for its limited functional diversity.

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Figure 1: Secondary structure, active site, and overall-fold SAXS envelope of the glmS ribozyme.
Figure 2: Large normal thio effects and lack of metal-ion rescue for the holoribozyme.
Figure 3: Large inverse thio effect and presence of metal-ion rescue for the aporibozyme.
Figure 4: Increasing concentrations of Mg2+ in the aporibozyme result in a diminished inverse thio effect for the RP thio substrate.
Figure 5: Active site structures from QM/MM optimizations for the glmS aporibozyme with Mg2+ closer to the RP position.
Figure 6: Multiple catalytic roles of the GlcN6P cofactor.

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Acknowledgements

We thank M. Been (Duke University Medical Center) for the generous gift of the plasmid containing the glmS ribozyme sequence, and we thank R. Gillilan and L. Pollack for help with SAXS experiments. We also thank E. Frankel and K. Leamy for assistance with fast hand-mixing reaction time points. Finally, we thank E. Frankel, R. Poudyal, L. Ritchey, and P. Thaplyal for helpful comments on revising the manuscript. This work was supported by US National Institutes of Health grant GM056207 (S.Z., D.R.S., and S.H.-S.) and US National Science Foundation grant CHE-1213667 (J.L.B. and P.C.B.). D.R.S. is supported as a member of the National Institutes of Health Chemistry-Biology Interface (training grant NRSA 1-T-32-GM070421). This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by the National Science Foundation. This work was based on research conducted at the Cornell High Energy Synchrotron Source (CHESS), which is supported by the National Science Foundation and the National Institutes of Health/National Institute of General Medical Sciences under NSF award DMR-0936384, in the Macromolecular Diffraction at CHESS (MacCHESS) facility, which is supported by award GM-103485 from the National Institutes of Health, through the National Institute of General Medical Sciences. We also thank the Penn State Proteomics and Mass Spectrometry Core Facility (University Park, PA) and the Penn State Genomics Core Facility (University Park, PA).

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Authors and Affiliations

  1. Department of Chemistry and Center for RNA Molecular Biology, Pennsylvania State University, University Park, Pennsylvania, USA

    Jamie L Bingaman & Philip C Bevilacqua

  2. Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Sixue Zhang, David R Stevens & Sharon Hammes-Schiffer

  3. X-ray Crystallography Facility, Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania, USA

    Neela H Yennawar

  4. Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania, USA

    Philip C Bevilacqua

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  1. Jamie L Bingaman
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Contributions

J.L.B. and P.C.B. designed experiments. J.L.B. performed the biochemistry experiments. J.L.B., N.H.Y., and P.C.B. collected and analyzed SAXS data. S.Z., D.R.S., and S.H.-S. designed calculations. S.Z. and D.R.S. performed the calculations. All authors wrote the manuscript.

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Correspondence to Sharon Hammes-Schiffer or Philip C Bevilacqua.

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Bingaman, J., Zhang, S., Stevens, D. et al. The GlcN6P cofactor plays multiple catalytic roles in the glmS ribozyme. Nat Chem Biol 13, 439–445 (2017). https://doi.org/10.1038/nchembio.2300

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