5lrt

Structure of the Deamidase-Depupylase Dop of the Prokaryotic Ubiquitin-like Modification Pathway in Complex with ADP and Phosphate

spinqualitylabelsall models 5lrt, resolution 1.85Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 5lrt is a 1 chain structure with sequence from Acidothermus cellulolyticus 11B. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.85Å Ligands: ADP, MG, NA, P6G, PEG, PGE, PO4 Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function DOP_ACIC1 Displays depupylase (DPUP) activity, removing conjugated Pup from target proteins; is thus involved in the recycling of Pup and may function similarly to deubiquitinases (DUBs) in eukaryotes to prevent or promote proteasomal degradation of certain proteins. Is also able to catalyze the deamidation of the C-terminal glutamine to glutamate in a variant of the prokaryotic ubiquitin-like protein Pup; however, since Pup from A.cellulolyticus possesses a C-terminal glutamate, this deamidase activity may be of no significance in vivo. Publication Abstract from PubMed Analogous to eukaryotic ubiquitination, proteins in actinobacteria can be post-translationally modified in a process referred to as pupylation, the covalent attachment of prokaryotic ubiquitin-like protein Pup to lysine side chains of the target protein via an isopeptide bond. Like in eukaryotes, an opposing activity counteracts the modification by specific cleavage of the isopeptide bond formed with Pup. However, the enzymes involved in pupylation and depupylation have evolved independently of ubiquitination and are related to the family of ATP-binding and hydrolyzing carboxylate-amine ligases of the glutamine synthetase type. Furthermore, the Pup ligase PafA and the depupylase Dop share close structural and sequence homology and have a common evolutionary history despite catalyzing opposing reactions. Here, we investigate the role played by the nucleotide in the active site of the depupylase Dop using a combination of biochemical experiments and X-ray crystallographic studies. We show that, although Dop does not turn over ATP stoichiometrically, the active site nucleotide species in Dop is ADP and inorganic phosphate rather than ATP, and that non-hydrolyzable analogs of ATP cannot support the enzymatic reaction. This finding suggests that the catalytic mechanism is more similar to the mechanism of the ligase PafA than previously thought and likely involves the transient formation of a phosphorylated Pup-intermediate. Evidence is presented for a mechanism where the inorganic phosphate acts as the nucleophilic species in amide bond cleavage and implications for Dop function are discussed. Depupylase Dop requires Inorganic Phosphate in the Active Site for Catalysis.,Bolten M, Vahlensieck C, Lipp C, Leibundgut M, Ban N, Weber-Ban E J Biol Chem. 2017 Jan 24. pii: jbc.M116.755645. doi: 10.1074/jbc.M116.755645. PMID:28119453 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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