Recently, a remarkable study showed that Mycobacteria have a distinct "ubiquitin-like" system in which a small protein, Pup, is transferred to the ε-amino groups of lysines in target proteins. These experiments also implicated a gene neighbor, the PafA protein, in this activity. How this was mediated was a mystery.Using sensitive sequence and structure analysis methods, we unified the PafA proteins to the glutamine synthetase (or carboxylate-amine/ammonia ligase) superfamily. In particular the PafA proteins are closer to the γ-glutamyl-cysteine synthetases. This unification provides a simple explanation for the reaction mechanism of Pupylation by PafA (the Pup ligase).
First the Pup ligase catalyzes an ATP-dependent phosphorylation of the γ-carboxylate of glutamate followed by ligation with the ε-amino group of lysines in target proteins with the formation of an amide linkage.
In Pups with a terminal glutamine instead of a glutamate (e.g. Mycobacterial Pup), the glutamine is first deamidated and converted to glutamate. Given the similar chemistry, we propose that this reaction too might be catalyzed by the Pup ligase. Our analysis suggests that pupylation is a bacterial innovation that emerged from proteins involved in amino acid (glutamine) and cofactor (glutathione) biosynthesis. The parallels with the ubiquitination system are striking in which the ubiquitin system evolved in bacteria from a system involved in cofactor (Moco) and amino acid (cysteine) biosynthesis. Thus the similiarities in pupylation and ubiquitination represent a remarkable case of convergent evolution.
Additional points of interest
- Pup is predicted to be a α-helical protein with an extended tail and is not related to ubiquitin.
- The pupylation system is present in most actinobacteria, and also sporadically in verrucomicrobia, nitrospirae, deltaproteobacteria and planctomycetes. In all cases both Pup and the Pup-ligase are immediate gene neighbors.
- Barring a few exceptions, gene neighborhoods reveal two paralogs of Pup ligases suggesting that they function as heterodimers. In species with only one copy, they would function as homodimers. Note Mycobacteria have two copies of PafA corresponding to genes Rv2097c and Rv2112c.
- Gene neighborhoods also reveal that the actinobacterial pupylation genes are neighbors of the archaeal-type proteasomal AAA+ ATPases and proteases (NTN hydrolase superfamily) in line with prior studies that in these bacteria pupylated proteins are targeted for degradation. However, this may not be always so. The Pup ligases of deltaproteobacteria and planctomycetes are remarkable in that they have 4 transmembrane helices inserted within the core domain and are also neighbors of membrane proteins. In these bacteria, the pupylation system might target membrane proteins.
- We also detected the prokaryotic homolog of the proteasomal chaperone PAC2 in the gene neighborhood of some actinobacterial Pupylation genes. This is the first report of a prokaryotic proteasomal chaperone and given the absence of other proteasomal chaperone subunits, it appears that PAC2 is the most ancient proteasomal chaperone (see the separate blog on PAC2).
- Could other members of this family catalyze analogous reactions? In this quest, we detected two other previously uncharacterized families of proteins that belong to the glutamine synthetase superfamily. However, their domain contexts and gene neighborhoods suggest that they may be involved in glutathione or related peptide secondary metabolites biosynthesis.
