Mycobacterium tuberculosis Prokaryotic Ubiquitin-like Protein-deconjugating Enzyme Is an Unusual Aspartate Amidase

Burns, Kristin E.; McAllister, Fiona E.; Schwerdtfeger, Carsten; Mintseris, Julian; Cerda-Maira, Francisca A.; Noens, Elke E.; Wilmanns, Matthias; Hubbard, Stevan R.; Melandri, Francesco; Ovaa, Huib; Gygi, Steven P.; Darwin, K. Heran

doi:10.1074/jbc.m112.384784

Cited by 20 publications

(22 citation statements)

References 41 publications

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“…In an earlier study it was shown that, during the cycle of catalysis, 18 O-water (H 2 18 O) is incorporated only into Pup and not into Dop (20). Furthermore, the authors showed that hydroxylamine can act as a nucleophile to form Pup-hydroxamate, indicating that during the Dop-catalyzed reaction an activated carbonyl must exist.…”

Section: Discussionmentioning

confidence: 99%

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Depupylase Dop Requires Inorganic Phosphate in the Active Site for Catalysis

Bolten

Vahlensieck

Lipp

et al. 2017

Journal of Biological Chemistry

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Edited by George N. DeMartinoAnalogous 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. As 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 ATPbinding 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 with substrate, 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.

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Section: Discussionmentioning

confidence: 99%

“…All Mg 2ϩ ions are coordinated in almost perfect octahedral symmetry. Notably, Asp-94, a residue previously shown to be important for activity (10,13) and proposed to form a mixed anhydride intermediate during catalysis (20), is coordinating Mg 2ϩ at the n1 position.…”

Section: Non-hydrolyzable Atp Analogs Cannot Support Dop Activity-mentioning

confidence: 99%

Depupylase Dop Requires Inorganic Phosphate in the Active Site for Catalysis

Bolten

Vahlensieck

Lipp

et al. 2017

Journal of Biological Chemistry

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“…Based on the crystal structure of a PafA D64N dimer solved with the C-terminally fused PupE38-E64 fragment reciprocally provided in trans (4), the C-terminal tail of Pup lines this groove and leads to the putative active site (Figure 1 d , upper panels). Pup E64 is positioned near the triphosphate chain of ATP and surrounded by PafA residues required for pupylation (4, 97), with analogous residues required for Dop activity (11, 97) (Figure 1 d , right panels).…”

Section: Pupylationmentioning

confidence: 99%

Prokaryotic Ubiquitin-Like Protein Modification

Maupin-Furlow

2014

Annu. Rev. Microbiol.

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Prokaryotes form ubiquitin (Ub)-like isopeptide bonds on the lysine residues of proteins by at least two distinct pathways that are reversible and regulated. In mycobacteria, the C-terminal Gln of Pup (prokaryotic ubiquitin-like protein) is deamidated and isopeptide linked to proteins by a mechanism distinct from ubiquitylation in enzymology yet analogous to ubiquitylation in targeting proteins for destruction by proteasomes. Ub-fold proteins of archaea (SAMPs, small archaeal modifier proteins) and Thermus (TtuB, tRNA-two-thiouridine B) that differ from Ub in amino acid sequence, yet share a common β-grasp fold, also form isopeptide bonds by a mechanism that appears streamlined compared with ubiquitylation. SAMPs and TtuB are found to be members of a small group of Ub-fold proteins that function not only in protein modification but also in sulfur-transfer pathways associated with tRNA thiolation and molybdopterin biosynthesis. These multifunctional Ub-fold proteins are thought to be some of the most ancient of Ub-like protein modifiers.

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“…In another study, Burns et al utilized an electrophilic trap consisting of Pup modified with a C-terminal glutamine mimic to identify a nucleophilic residue in Dop. Labeling of aspartate 95, an atypical nucleophile for a protease, classified Dop as an unusual amidase whereby the deamidation or depupylation reaction proceeds via an anhydride intermediate (61). Although this model is supported by the crystal structure, further experimentation is necessary to definitively prove this unusual protease activity.…”

Section: Depupylationmentioning

confidence: 99%

The Pup-Proteasome System of Mycobacteria

Bode

Darwin

2014

Microbiol Spectr

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Proteasomes are ATP-dependent, barrel-shaped proteases found in all three domains of life. In eukaryotes, proteins are typically targeted for degradation by posttranslational modification with the small protein ubiquitin. In 2008, the first bacterial protein modifier, Pup (prokaryotic ubiquitin-like protein) was identified in Mycobacterium tuberculosis (Mtb). Functionally analogous to ubiquitin, conjugation with Pup serves as a signal for degradation by the mycobacterial proteasome. Proteolysis-dependent and -independent functions of the Mtb proteasome are essential for virulence of this successful pathogen. In this chapter we describe the discovery of the proteasome as a key player in tuberculosis pathogenesis and the biology and biochemistry of the Pup-proteasome system.

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Mycobacterium tuberculosis Prokaryotic Ubiquitin-like Protein-deconjugating Enzyme Is an Unusual Aspartate Amidase

Cited by 20 publications

References 41 publications

Depupylase Dop Requires Inorganic Phosphate in the Active Site for Catalysis

Depupylase Dop Requires Inorganic Phosphate in the Active Site for Catalysis

Prokaryotic Ubiquitin-Like Protein Modification

The Pup-Proteasome System of Mycobacteria

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