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Structure and function of the bacterial decapping enzyme NudC

Nature Chemical Biology volume 12, pages 730–734 (2016)Cite this article

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Abstract

RNA capping and decapping are thought to be distinctive features of eukaryotes. The redox cofactor NAD was recently discovered to be attached to small regulatory RNAs in bacteria in a cap-like manner, and Nudix hydrolase NudC was found to act as a NAD-decapping enzyme in vitro and in vivo. Here, crystal structures of Escherichia coli NudC in complex with substrate NAD and with cleavage product NMN reveal the catalytic residues lining the binding pocket and principles underlying molecular recognition of substrate and product. Biochemical mutation analysis identifies the conserved Nudix motif as the catalytic center of the enzyme, which needs to be homodimeric, as the catalytic pocket is composed of amino acids from both monomers. NudC is single-strand specific and has a purine preference for the 5′-terminal nucleotide. The enzyme strongly prefers NAD-linked RNA (NAD–RNA) over NAD and binds to a diverse set of cellular RNAs in an unspecific manner.

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Figure 1: Crystal structure E. coli NudC in complex with bound NAD.
Figure 2: Crystal structure of E. coli NudC in complex with bound NMN.
Figure 3: Mutagenesis of NudC.
Figure 4: RNA mutagenesis.
Figure 5: Comparison of the active site conformation in decapping enzymes.

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Acknowledgements

We are grateful to L. Obenauer (Heidelberg University) for assistance with NGS data analysis, L. Kiss for experimental assistance and to W. Shi at beamline X29A at the Brookhaven National Laboratory for support in diffraction data collection. A.J. is supported by the German Research Foundation (DFG SPP 1784, grant JA 794/10-1) and by the Baden-Württemberg Stiftung (BWST-NCRNA-045). The structural research was supported by funds from US National Institutes of Health (NIH) grant GM104962 to D.J.P. and the Thousand Young Talent Program of China and the Chinese Academy of Sciences to J.D.

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  1. Katharina Höfer and Sisi Li: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany

    Katharina Höfer, Florian Abele, Jens Frindert, Jasmin Schlotthauer, Julia Grawenhoff & Andres Jäschke

  2. Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Sisi Li & Dinshaw J Patel

  3. Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    Jiamu Du

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  1. Katharina Höfer
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Contributions

K.H., S.L., D.J.P., and A.J. designed research; S.L. and J.D. performed the crystallographic investigation; K.H., F.A., J.F., J.S., and J.G. carried out the functional studies on NudC; all authors analyzed data; K.H., S.L., J.F., J.D., D.J.P., and A.J. wrote the paper.

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Correspondence to Dinshaw J Patel or Andres Jäschke.

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Supplementary Results, Supplementary Figures 1–8 and Supplementary Tables 1 and 2. (PDF 3030 kb)

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Höfer, K., Li, S., Abele, F. et al. Structure and function of the bacterial decapping enzyme NudC. Nat Chem Biol 12, 730–734 (2016). https://doi.org/10.1038/nchembio.2132

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