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Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes

Nature Structural & Molecular Biology volume 16, pages 647–651 (2009)Cite this article

Abstract

In analogy to ubiquitin in eukaryotes, the bacterial protein Pup is attached to lysine residues of substrate proteins, thereby targeting them for proteasomal degradation. It has been proposed that, before its attachment, Pup is modified by deamidation of its C-terminal glutamine to glutamate. Here we have identified Dop (locus tag Rv2112) as the specific deamidase of Pup in Mycobacterium tuberculosis. Deamidation requires ATP as a cofactor but not its hydrolysis. Furthermore, we provide experimental evidence that PafA (locus tag Rv2097) ligates deamidated Pup to the proteasomal substrate proteins FabD and PanB. This formation of an isopeptide bond requires hydrolysis of ATP to ADP, suggesting that deamidated Pup is activated for conjugation via phosphorylation of its C-terminal glutamate. By combining these enzymes, we have reconstituted the complete bacterial ubiquitin-like modification pathway in vitro, consisting of deamidation and ligation steps catalyzed by Pup deamidase (Dop) and Pup ligase (PafA).

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Figure 1: Pup interacts with PafA and Dop, two homologous but distinct enzymes.
Figure 2: Dop deamidates the C-terminal glutamine of Pup.
Figure 3: Dop does not hydrolyze ATP during deamidation of Pup.
Figure 4: PafA is sufficient to conjugate deamidated Pup to a substrate protein.
Figure 5: Pup is coupled to substrates by separate deamidation and conjugation steps.

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Acknowledgements

We thank the staff at the Functional Genomics Center Zurich (FGCZ) for MS, A. Geerlof (European Molecular Biology Laboratory, Hamburg) for the pMyC expression vector, the Richmond laboratory (ETH Zürich) for the His6-Thioredoxin-TEV fusion vector and P. Sander (University of Zurich) for the mycobacterial strains. This work was supported by the Swiss National Science Foundation (SNF), the National Center for Excellence in Research (NCCR) Structural Biology program of the SNF, an ETH research grant and a Kekulé fellowship by the 'Fonds der Chemischen Industrie' to F.S.

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  1. ETH Zurich, Institute of Molecular Biology & Biophysics, Zurich, Switzerland

    Frank Striebel, Frank Imkamp, Markus Sutter, Martina Steiner, Azad Mamedov & Eilika Weber-Ban

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Correspondence to Eilika Weber-Ban.

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Striebel, F., Imkamp, F., Sutter, M. et al. Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes. Nat Struct Mol Biol 16, 647–651 (2009). https://doi.org/10.1038/nsmb.1597

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