Unraveling the biochemistry and provenance of pupylation: a prokaryotic analog of ubiquitination

Abstract

Recently Mycobacterium tuberculosis was shown to possess a novel protein modification, in which a small protein Pup is conjugated to the epsilon-amino groups of lysines in target proteins. Analogous to ubiquitin modification in eukaryotes, this remarkable modification recruits proteins for degradation via archaeal-type proteasomes found in mycobacteria and allied actinobacteria. While a mycobacterial protein named PafA was found to be required for this conjugation reaction, its biochemical mechanism has not been elucidated. Using sensitive sequence profile comparison methods we establish that the PafA family proteins are related to the γ-glutamyl-cysteine synthetase and glutamine synthetase. Hence, we predict that PafA is the Pup ligase, which catalyzes the ATPdependent ligation of the terminal γ-carboxylate of glutamate to lysines, similar to the above enzymes. We further discovered that an ortholog of the eukaryotic PAC2 (e.g. cg2106) is often present in the vicinity of the actinobacterial Pup-proteasome gene neighborhoods and is likely to represent the ancestral proteasomal chaperone. Pup-conjugation is sporadically present outside the actinobacteria in certain lineages, such as verrucomicrobia, nitrospirae, deltaproteobacteria and planctomycetes, and in the latter two lineages it might modify membrane proteins. Reviewers: This article was reviewed

A distinct structural region of the prokaryotic ubiquitin‐like protein (Pup) is recognized by the N‐terminal domain of the proteasomal ATPase Mpa

“…The conjugation reaction consists of two steps: First, Dop (deamidase of Pup) deamidates the C-terminal glutamine of Pup, converting its C-terminus from GGQ to GGE [15]. This renders Pup competent for conjugation to substrates by the Pup-ligase PafA [15], the function of which had also been predicted by the absence of pupylated substrates in a pafA-knockout strain [14] and bioinformatic analysis [16]. Interestingly, in many organisms the Pup protein ends with GGE ( Fig.…”

Allosteric Transitions Direct Protein Tagging by PafA, the Prokaryotic Ubiquitin-like Protein (Pup) Ligase

“…Specifically, such mutants are sensitized to reactive nitrogen intermediates, antimicrobial compounds that are released into phagosomes by macrophages (11,12). Noteworthy, the vast majority of Pup/proteasome-containing bacteria are nonpathogenic, suggesting a fundamental role of this proteolytic pathway in these species (13,14). Despite apparent functional similarity between Pup and ubiquitin, these degradation tags are not homologous.…”

“…The Pup ligase, an enzyme termed PafA (proteasomal accessory factor A), both activates and conjugates Pup to protein substrates in a two-step reaction that is typical of glutamine synthetases and ␥-glutamyl cysteine synthetases (2,14,19). Indeed, PafA was identified as a distant homologue of this group of enzymes (14). The first step in the PafA-catalyzed reaction is the phosphorylation of Pup on the ␥-carboxylate of its C-terminal glutamate.…”

“…However, the enzymatic mechanism of Pup activation and conjugation is altogether different from that of ubiquitin (1,20). The Pup ligase, an enzyme termed PafA (proteasomal accessory factor A), both activates and conjugates Pup to protein substrates in a two-step reaction that is typical of glutamine synthetases and ␥-glutamyl cysteine synthetases (2,14,19). Indeed, PafA was identified as a distant homologue of this group of enzymes (14).…”

Mycobacterial Ubiquitin-like Protein Ligase PafA Follows a Two-step Reaction Pathway with a Phosphorylated Pup Intermediate

“…Bioinformatic investigations have shown that both PafA and Dop belong to the carboxylate-amine/ammonia ligase superfamily, a group that contains glutamine synthetase, ␥-glutamylcysteine synthetase, and the amidotransferase Gat-CAB (18). The reaction mechanism for all of these family members is thought to follow a two-step reaction pathway in which the ␥-glutamyl carboxylate is phosphorylated using ATP as the phosphate donor, followed by nucleophilic attack by either ammonia (glutamine synthetase, GatCAB) or the ␣-amino group of cysteine (␥-glutamylcysteine synthetase) (19 -24).…”