A mystery pathway in prokaryotes

Computational studies of proteins have greatly contributed to our understanding of the biology of a species or a system.  In many instances, computational analyses have solved tricky biochemical problems (e.g. the biochemistry of pupylation), or have uncovered unexpected systems or pathways (e.g. the prokaryotic cognates of the eukaryotic ubiquitin pathway), or solved long-standing mysteries (e.g. the principal transcription factors of apicomplexa), or clarified difficult evolutionary problems (e.g. the extent of lateral transfer between prokaryotes, the evolutionary origins of the AID/APOBEC deaminases). Yet there are instances, when the biochemistry of most parts of a system are easily identifiable, but the biology remains an unsolved puzzle. Recently, we uncovered one such widespread system present in most lineages of proteobacteria, actinobacteria, spirochaetes, cyanobacteria, chlamydiae and chloroflexi and also some crenarchaea. As the system is present in Mycobacterium tuberculosis, we shall use the Mycobacterial gene names  as representative identifiers. The basic system consists of

1. Rv2410c (DUF403 in Pfam 25) : An alpha-helical protein,called Alpha-E  that contains an internal duplication with each repeat possessing conserved ER motifs. Click here to access a multiple alignment.
2. Rv2411c (split as DUF404+DUF407 in Pfam 25): A circularly permuted peptide ligase of the ATP-grasp fold.
3. Rv2409c, Rv2569c: Transglutaminases that could serve either as a peptidase or a classical transglutaminase.
4. Rv2568c (DUF2248 in Pfam 25): A metallopeptidase-family peptidase.
5. Rv2567: An inactive circularly permuted ATP-grasp fused to the Alpha-E domain.
6. Rv2566 (Transglut+DUF2126 in Pfam 25): A transglutaminase fused to a circularly permuted peptide ligase of the COOH-NH2 ligase superfamily.
7. Some species additionally contain an NTN hydrolase related to the  proteasomal peptidase (called Anbu in one study) in the gene neighborhoods (not  Mycobacterium) and amidotransferases of the GAT-I family.  Click here to access all operons.

Thus, these systems together include two active peptide ligases, 5 distinct types peptidase-like proteins (2 transglutaminases, Zincin-like metallopeptidase, the GAT-I domain and a NTN peptidase) , the mystery Alpha-E  protein and an inactive peptide ligase that may be fused to the mystery Alpha-E domain. In any case all systems minimally contain at least one peptide ligase, the Alpha-E protein and one peptidase-like domain. The only evidence for its biological context comes from experiments in Pseudomonas putida where the transglutaminase is highly expressed upon nitrogen starvation. Several protein/peptide conjugation systems contain  peptide ligases (e.g. the ubiquitin transferring enzymes, the Pup ligases) as well as deconjugating emzymes (e.g. JAB deubiquitinase and Dop depupylase) in the same gene context (For a comprehensive set of examples, read our paper on amidoligases).
However, assembling the pieces of the puzzle together, we can be sure of a few things

1. This is not involved in amino acid or glutathione biosynthesis. The species containing this system typically have intact pathways for glutathione or amino acid biosynthesis. Also there are no other genes suggestive of metabolic function in the neighborhood.
2. It is not involved in the biosynthesis of a distinctive secondary metabolite such as an antibiotic or siderophore, for it lacks characteristic associations seen in these systems (see examples in our study of such systems).
3. There is no evidence of a small protein that is conjugated to a target as in ubiquitination or pupylation.

Gene neighborhoods of the novel system described in this post

Thus the system appears to be a novel peptide transfer/peptidase system with the Alpha-E protein playing a central role.  We postulate that the ATP-grasp and COOH-NH2 ligase in this system catalyze two distinct peptide bond formations. It is tempting to speculate that the Alpha-E protein with the highly conserved ER motifs serve as a substrate for elongation of a peptide via the gammacarboxylate of its side chain. This proposal is consistent with the use of glutamate side chains as substrates in eukaryotic proteins such as tubulin by peptide tagging ATP-grasp enzymes.The presence of two peptidase genes in most of these operons suggests that two successive peptidase reactions are necessary for removal of the peptide product.
 Alternatively, the transglutaminase superfamily protein might indeed function in cross-linking the peptide to lysine side chains or other amino groups. Thus, the weight of the contextual evidence supports a role for this widespread conserved gene-neighborhood in peptide synthesis; the resulting peptide could be added as a tag to the unique Alpha-E protein in this system.Such a tag could either regulate the assembly of complexes of the alpha-E domain protein via cross-linking or its interactions (e.g. as in tubulin) or serve as an amino acid storage mechanism. Yet, as you can see, certain details of this interesting pathway are in need of further investigation, but its widespread presence suggests that an important and exciting piece of biology awaits creative experimentalists...

The fellowship of the RING: Bacteria have them too!

If there is one lesson to learn from comparative genomics, it is to never underestimate the bacteria. Bacteria are verily the engines of protein diversity and have provided some of the most remarkable insights on the origins of various pathways and systems. In our next release on the ubiquitin-mediated signaling/ tagging/ protein turnover pathway, we address the origins and roles of several treble clef domains, including the RING finger, found in the eukaryotic ubiquitin pathway. This study also answers some tricky questions raised in a previous post.

Q1. Do any species have the entire complement of both the ubiquitin and pupylation based protein turnover/tagging system?

Indeed, some actinobacteria such as Frankia and planctomycetes such as Pirellula staleyi contain both the entire complement of the ubiquitin system and pupylation. The list of species with both systems is only expected to grow as more such genomes are sequenced. The discovery of the Frankia RING finger was a bit complicated. It turns out that the RING finger is encoded in the opposite strand to the sequence that is submitted to the database and whats more the RING is sandwiched between an E1 and E2. For details, peruse our supplement.

Q2. If so, how is the labor of protein turnover divided between pupylation and ubiquitination?

This far we can only make a reasonable guess, but this is where experiments will reveal more. There is, however, a curious aspect to the prokaryotic RING-finger containing Ub-systems. Many of the core Ub-pathway proteins have transmembrane (TM) helices. In Frankia, both the JAB deubiquitinase and the RING are fused to transmembrane helices. In Pirellula, the RING finger while containing a TM helix is also fused to a distinct domain prototyped by DUF3137, a potential solute sensor. This suggests that a major fraction of the prokaryotic RING domains might have functions related to either regulation or modification of membrane-associated proteins. While the pupylation system of actinobacteria is cytoplasmic, the Pirellula Pup ligase, like other planctomycete versions, has 4 transmembrane helices inserted within the core domain, suggesting that in some species, pupylation has a strong membrane component. Study the architectures and associations of these Ub-systems here

Q3. In light of the discovery in Caldiarchaeum, what can we say about the origins of the eukaryotic Ub-system?

The Caldiarchaeum Ub-system is remarkable in that each of its Ub-system components is very closely related in sequence and structure to the corresponding eukaryotic version. For example, the E1 protein of this archaeon contains a C-terminal Ub-fold UFD domain (that was only detected in eukaryotes to date). However, this far no other archaeon contains a complete complement of the Ub-system. Further bacterial versions of the complete Ub-system are also sporadically distributed, suggesting a strong component of lateral transfer in the dissemination of this system across prokaryotes. Therefore, we cannot be certain if the eukaryotic Ub-system emerged from a Caldiarchaeum-like system in the archaeal symbiont during eukaryogenesis. Indeed, such systems might be present in as yet un-sampled bacteria suggesting that it is not unlikely that eukaryotes acquired such a system from the primary bacterial symbiont or even via an independent lateral transfer of the operon from yet another prokaryote.

Based on this and our previous study on the ubiquitin system, we can now confidently state that systems resembling eukaryotic Ub-conjugation systems were put together to different degrees in prokaryotes during the diversification of various biosynthetic and regulatory pathways. With regards to the proteasomal association, while the core proteasomal apparatus is of archaeal origin, it is also present in various bacteria of which some possess the complete core Ub-system. Hence, it is possible that this connection between the Ub system and the proteasome developed either in bacteria or archaea, and was merely retained in eukaryotes which vertically inherited their core proteasomal complex from the archaea.

Wait.. there much more to this story. Feel free to access the paper here and browse through the extensive supplement.

Where Pupylation and Ubiquitination co-occur, who does what?

Recently, the authors sequencing the archaeon Caldiarchaeum unearthed a remarkable operon with the entire core of the ubiquitin system containing genes encoding Ubiquitin, E1,E2,E3 and the deubiquitinase of the JAB family(also called MPN1) (Click here to read). These proteins are remarkable in that they share several sequence features with their eukaryotic counterparts. Further, the archaeon, like all members of its clade, contains the archaeo-eukaryotic proteasomal degradation system, suggesting the presence of the basic complement of the ubiquitin-based protein turnover system.

Great progress has also been made on the biochemistry of the other peptide-tag based protein turnover system; the pupylation pathway. A large number of substrates have now been reported and we also know that the Pup-ligase (PafA) paralog (also called Dop) which in some species deamidates glutamine, is a depupylase (Click here to read).

The consequence of these studies raises some interesting possibilities and questions.

1. Do any species have the entire complement of both the ubiquitin and pupylation based protein turnover/tagging system?

2. If so, how is the labor of protein turnover divided between pupylation and ubiquitination?

3. In light of the discovery in Caldiarchaeum, what can we say about the origins of the Ub-system?

Answers to these questions are in the following post. Click here to access the answers.

What is the biochemistry of Pupylation?

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.

For more details, you can read the open access version of the paper. Click here to access it. For latest updates on pupylation click here