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

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).

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

“…5). As we demonstrated previously [15], full-length Mpa binds to immobilized Pup. The interdomain and interdomain-AAA fragments are not recruited by Pup-decorated beads.…”

“…Pup interacts with two processing enzymes, Dop and PafA, that deamidate and couple, respectively, its C-terminus to substrate proteins [15]. This marks the substrates for recognition by the proteasomal chaperone Mpa and degradation by the 20S proteasome [12,14].…”

“…Pup was expressed and purified as described [15]. 15 N-labeled protein was produced by growing the cells in M9 minimal medium supplemented with 99% 15 N ammonium chloride and for 13 C-labeling with 99% [ 13 C 6 ] glucose.…”

“…A cysteine variant of Pup (PupS-1C) was obtained by changing the N-terminal Gly-Ser to Gly-Cys by site-directed-mutagenesis [15]. PupS-1C was labeled with fluorescein-5-maleimide (Perbio Science) and purified on a MonoQ column (GE Healthcare) [28].…”

“…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.…”