EpmA (also called GenX, PoxA or YjeA) is a cytoplasmic, homodimeric paralog of class II lysyl-tRNA synthetase that has lost the N-terminal anticodon-binding domain and does not aminoacylate tRNA. Instead it uses its retained class II catalytic core to activate (R)-beta-lysine, made by the aminomutase EpmB, with ATP as a beta-lysyl-adenylate, and transfers the beta-lysyl group to the epsilon-amino group of Lys34 of translation elongation factor P (EF-P), a protein that mimics the shape of tRNA and docks on EpmA much as tRNA(Lys) docks on LysRS. (R)-beta-lysine is preferred about 100-fold over (S)-beta-lysine or L-alpha-lysine. The beta-lysylated residue is then hydroxylated by EpmC. Modified EF-P is the active form that relieves ribosome stalling at polyproline stretches, so loss of EpmA reduces expression of polyproline-containing proteins such as the acid-stress regulator CadC, causing slow growth at low pH, loss of lysine decarboxylase induction and, in Salmonella, pleiotropic stress-sensitivity and virulence defects.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000049 tRNA binding | IBA GO_REF:0000033 | REMOVE | Summary: tRNA binding, propagated from the lysyl-tRNA synthetase ancestral node PTN000502669 (rat KARS1 as donor). Reason: EpmA belongs to PANTHER PTHR42918 (lysyl-tRNA synthetase) but to the divergent subfamily SF6, which lost the N-terminal anticodon-binding (OB-fold) domain that LysRS uses to bind tRNA. Roy et al. show that PoxA does not recognize tRNA and cannot aminoacylate tRNA(Lys); its macromolecular substrate is the tRNA mimic EF-P. The function at the LysRS node is real for LysRS, but the propagation into the EpmA subfamily is wrong: the subfamily has demonstrably diverged, so this row is removed. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN000502669 Β· PANTHER:PTN000502669 SUPPORTS SOURCE BUT NOT TARGET LysRS ancestral node; tRNA binding is right for the LysRS clade, but the EpmA subfamily (PTHR42918:SF6) lost the anticodon-binding domain and tRNA recognition. RGD:1359653 Β· rat Kars1 SUPPORTS SOURCE BUT NOT TARGET Canonical lysyl-tRNA synthetase; its tRNA binding does not transfer to EpmA. Supporting Evidence: PMID:21841797 One notable exception is PoxA, a paralog of lysyl-tRNA synthetase (LysRS), which does not recognize tRNA but instead post-translationally modifies elongation factor P (EF-P) PMID:21841797 Elimination of the amino-terminal anticodon-binding domain of LysRS would have substantially reduced affinity for tRNA file:ECOLI/epmA/epmA-uniprot.txt Was originally suggested to be a tRNA synthase, however its lack of an anticodon-binding domain made this highly unlikely. |
| GO:0000166 nucleotide binding | IEA GO_REF:0000002 | MODIFY | Summary: Nucleotide binding, from the class II aminoacyl-tRNA synthetase core signatures. Reason: Correct but general. The nucleotide EpmA binds is ATP, its co-substrate (Km 206 uM), at binding sites mapped in the class II core; EpmA already carries the child GO:0005524 ATP binding, which is the replacement. Proposed replacements: ATP binding Supporting Evidence: file:ECOLI/epmA/epmA-uniprot.txt KM=206 uM for ATP |
| GO:0004812 aminoacyl-tRNA ligase activity | IEA GO_REF:0000002 | REMOVE | Summary: Aminoacyl-tRNA ligase activity, mapped from the class II aaRS core domain signature (IPR004364). Reason: The InterPro2GO mapping assumes every class II aaRS core domain charges a tRNA. EpmA keeps the core and the amino-acid activation half-reaction, but transfers the activated beta-lysine to a protein, not to tRNA, and cannot aminoacylate tRNA(Lys). The mapping is demonstrably wrong for this subfamily. Supporting Evidence: PMID:21841797 PoxA cannot aminoacylate tRNALys, indicating that while it is structurally similar to LysRS its activity is different from that of a canonical aaRS file:ECOLI/epmA/epmA-uniprot.txt Cannot ligate lysine to any tRNA. |
| GO:0004824 lysine-tRNA ligase activity | IBA GO_REF:0000033 | REMOVE | Summary: Lysine-tRNA ligase activity, propagated from the LysRS ancestral node (E. coli LysS and LysU, human KARS1, yeast KRS1/MSK1 and others). Reason: The donors are bona fide lysyl-tRNA synthetases, including the two E. coli LysRS paralogs LysS (P0A8N3) and LysU (P0A8N5), which carry out tRNA(Lys) charging in this organism. EpmA sits in the divergent SF6 subfamily: it cannot ligate lysine to any tRNA, and its activity is orthogonal to that of LysRS (beta-lysine onto EF-P). The IBA should have been blocked by a loss at the EpmA subfamily node. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN000502669 Β· PANTHER:PTN000502669 SUPPORTS SOURCE BUT NOT TARGET LysRS ancestral node; EpmA subfamily (PTHR42918:SF6) diverged to protein lysylation. UniProtKB:P0A8N3 Β· E. coli LysS SUPPORTS SOURCE BUT NOT TARGET Genuine LysRS paralog in the same organism; performs the tRNA(Lys) charging EpmA cannot. UniProtKB:P0A8N5 Β· E. coli LysU SUPPORTS SOURCE BUT NOT TARGET Heat-inducible LysRS paralog; genuine lysine-tRNA ligase. Supporting Evidence: PMID:21841797 PoxA cannot aminoacylate tRNALys, indicating that while it is structurally similar to LysRS its activity is different from that of a canonical aaRS PMID:21841797 EF-P was efficiently functionally modified with (R)-Ξ²-lysine but not (S)-Ξ²-lysine or genetically encoded Ξ±-amino acids, indicating that PoxA has evolved an activity orthogonal to that of the canonical aminoacyl-tRNA synthetases file:ECOLI/epmA/epmA-uniprot.txt Cannot ligate lysine to any tRNA. |
| GO:0004824 lysine-tRNA ligase activity | IEA GO_REF:0000002 | REMOVE | Summary: Lysine-tRNA ligase activity, mapped from InterPro IPR004525 (EpmA) and IPR018149 (LysRS class II C-terminal region). Reason: Wrong for this gene. IPR004525 is the EpmA family entry itself, whose members ligate beta-lysine to EF-P rather than lysine to tRNA, so mapping it to lysine-tRNA ligase activity is an InterPro2GO error; EpmA cannot ligate lysine to any tRNA. Same verdict as the IBA row. Supporting Evidence: PMID:21841797 PoxA cannot aminoacylate tRNALys, indicating that while it is structurally similar to LysRS its activity is different from that of a canonical aaRS file:ECOLI/epmA/epmA-uniprot.txt Cannot ligate lysine to any tRNA. |
| GO:0005515 protein binding | IPI PMID:15690043 Interaction network containing conserved and essential prote... | REMOVE | Summary: Co-purification with AceE (pyruvate dehydrogenase E1) in a large-scale affinity-purification mass spectrometry screen. Reason: Bare protein binding is uninformative and is removed under the project protein-binding policy. The AceE association comes from a high-throughput pull-down without functional follow-up; the biologically meaningful EpmA partner is its substrate EF-P, which is captured by the lysyltransferase activity. Supporting Evidence: PMID:15690043 we have targeted DNA cassettes into the E. coli chromosome to create carboxy-terminal, affinity-tagged alleles of 1,000 open reading frames |
| GO:0005524 ATP binding | IEA GO_REF:0000120 | ACCEPT | Summary: ATP binding by the class II catalytic core, used to form the beta-lysyl-adenylate intermediate. Reason: ATP is a measured co-substrate (Km 206 uM) and the structure was solved with a lysyl-adenylate analog; ATP-binding residues are mapped in the class II motifs. Binding ATP is an integral part of the core ligase reaction. Supporting Evidence: file:ECOLI/epmA/epmA-uniprot.txt KM=206 uM for ATP file:ECOLI/epmA/epmA-uniprot.txt lysine produced by EpmB, forming a lysyl-adenylate |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | MODIFY | Summary: Active in the cytoplasm, propagated from the LysRS node; EpmA itself is among the descendant evidences. Reason: The location is right (unlike the tRNA terms from the same node, it holds for EpmA, which has no signal peptide or membrane segment and acts on cytoplasmic EF-P), but in a bacterium the soluble cytoplasm is the cytosol, where EpmA has been detected directly (IDA, PMID:18304323). The replacement is the more specific GO:0005829 cytosol that EpmA already carries. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000502669 Β· PANTHER:PTN000502669 SUPPORTS TRANSFER Cytoplasmic location is shared by LysRS and EpmA; only granularity is refined. UniProtKB:P0A8N7 Β· EpmA SUPPORTS TRANSFER Target's own cytosol IDA is among the descendant evidences, as expected. Proposed replacements: cytosol Supporting Evidence: PMID:18304323 we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100 |
| GO:0005737 cytoplasm | IEA GO_REF:0000002 | MODIFY | Summary: Cytoplasm, from InterPro family mappings. Reason: Correct but general; EpmA is a soluble enzyme detected in the cytosolic fraction (IDA), so the replacement is GO:0005829 cytosol, which EpmA already carries. Same action as the IBA row. Proposed replacements: cytosol Supporting Evidence: PMID:18304323 we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100 |
| GO:0005829 cytosol | IDA PMID:18304323 Protein abundance profiling of the Escherichia coli cytosol. | ACCEPT | Summary: Detected in the cytosolic fraction by proteomic abundance profiling of E. coli MC4100. Reason: Fractionation proteomics places EpmA in the cytosol, consistent with a soluble enzyme without targeting signals whose substrate EF-P is cytosolic. The protein list is in supplementary data not in the cache; the curator's IDA is accepted. Supporting Evidence: PMID:18304323 we identified 1103 proteins from the cytosolic fraction of the Escherichia coli strain MC4100 |
| GO:0006418 tRNA aminoacylation for protein translation | IEA GO_REF:0000002 | REMOVE | Summary: tRNA aminoacylation for protein translation, mapped from the class II aaRS core signature. Reason: EpmA does not charge any tRNA; the class II core in this subfamily was repurposed to aminoacylate a protein (EF-P Lys34). The InterPro2GO mapping is wrong for EpmA. Supporting Evidence: PMID:21841797 PoxA cannot aminoacylate tRNALys, indicating that while it is structurally similar to LysRS its activity is different from that of a canonical aaRS PMID:20729861 it specifically aminoacylates EF-P with lysine |
| GO:0006430 obsolete lysyl-tRNA aminoacylation | IBA GO_REF:0000033 | REMOVE | Summary: Lysyl-tRNA aminoacylation, propagated from the LysRS ancestral node. Reason: Over-propagation from genuine LysRS donors (including E. coli LysS and LysU) into the divergent EpmA subfamily. EpmA cannot aminoacylate tRNA(Lys); the process it takes part in is post-translational modification of EF-P. The term is also obsolete in GOA. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN000502669 Β· PANTHER:PTN000502669 SUPPORTS SOURCE BUT NOT TARGET LysRS ancestral node; tRNA charging was lost in the EpmA subfamily. UniProtKB:P0A8N3 Β· E. coli LysS SUPPORTS SOURCE BUT NOT TARGET Genuine LysRS in the same organism. UniProtKB:P9WFU7 Β· M. tuberculosis LysS SUPPORTS SOURCE BUT NOT TARGET Genuine LysRS. Supporting Evidence: PMID:21841797 PoxA cannot aminoacylate tRNALys, indicating that while it is structurally similar to LysRS its activity is different from that of a canonical aaRS PMID:21841797 Ξ²-lysine was also a potent inhibitor of Ξ±-lysine modification of EF-P, consistent with this reaction being orthogonal to tRNA aminoacylation by LysRS |
| GO:0006430 obsolete lysyl-tRNA aminoacylation | IEA GO_REF:0000002 | REMOVE | Summary: Lysyl-tRNA aminoacylation, mapped from InterPro IPR004525 (EpmA) and IPR018149. Reason: Same verdict as the IBA row; mapping the EpmA family entry itself to lysyl-tRNA aminoacylation is an InterPro2GO error, because EpmA cannot ligate lysine to any tRNA. Supporting Evidence: file:ECOLI/epmA/epmA-uniprot.txt Cannot ligate lysine to any tRNA. |
| GO:0016880 acid-ammonia (or amide) ligase activity | IEA GO_REF:0000104 | MODIFY | Summary: Acid-ammonia (or amide) ligase activity, from the HAMAP/UniRule EpmA rule. Reason: The essence is right: EpmA is an ATP-dependent ligase that forms an amide (isopeptide) bond between the carboxyl of (R)-beta-lysine and the epsilon-amino group of EF-P Lys34. But the acid-ammonia class does not describe a protein-modifying reaction, and GO has an exact term for this reaction: GO:0052868 protein-lysine lysyltransferase activity, whose definition is the RHEA:83435 equation and which EpmA already carries by IDA. This is a lateral replacement; GO:0052868 sits under aminoacyltransferase activity, not under this ligase term. Proposed replacements: protein-lysine lysyltransferase activity Supporting Evidence: file:ECOLI/epmA/epmA-uniprot.txt Reaction=D-beta-lysine + L-lysyl-[protein] + ATP |
| GO:0042803 protein homodimerization activity | IDA PMID:20729861 A paralog of lysyl-tRNA synthetase aminoacylates a conserved... | KEEP AS NON CORE | Summary: EpmA forms a homodimer, as shown by the crystal structures and supported by the crystallization study (UniProt subunit, experimental evidence from PMID:20729861 and PMID:20823541). Reason: Homodimerization is a structural property inherited from the class II aaRS fold, whose active site is built at the dimer; it is real but is not the evolved function. The cached Yanagisawa paper is abstract-only, so the curated UniProt subunit line tied to it is quoted and the curator's IDA is kept. Supporting Evidence: file:ECOLI/epmA/epmA-uniprot.txt SUBUNIT: Homodimer. |
| GO:0043687 post-translational protein modification | RCA PMID:20070887 Predicting the pathway involved in post-translational modifi... | ACCEPT | Summary: Post-translational protein modification, from the comparative-genomics prediction that YjeA and YjeK modify EF-P (PMID:20070887), since confirmed biochemically (PMID:20729861, PMID:21841797, PMID:22128152). Reason: EpmA performs the covalent step itself: it ligates (R)-beta-lysine to Lys34 of fully translated, free EF-P, which is what this term covers (modification after release from the ribosome). This differs from EpmB, whose identical RCA row was marked over-annotated because EpmB only supplies the free donor and does not modify the protein; EpmA is the enzyme that does the work, so the term is core here. No more specific GO process exists for EF-P beta-lysylation. Supporting Evidence: PMID:20070887 we predict that the YjeA and YjeK families of proteins are involved in EF-P modification in a subset of bacterial species PMID:20729861 it specifically aminoacylates EF-P with lysine PMID:22128152 indicating that both enzymes, YjeA and YjeK, are required for Ξ²-lysylation of EF-P |
| GO:0052868 protein-lysine lysyltransferase activity | IDA PMID:20729861 A paralog of lysyl-tRNA synthetase aminoacylates a conserved... | ACCEPT | Summary: EpmA lysylates EF-P in vitro (PMID:20729861); the physiological donor was later shown to be (R)-beta-lysine (PMID:21841797). Reason: This is the core activity. GO:0052868 is defined by exactly the EpmA reaction (D-beta-lysine + L-lysyl-[protein] + ATP to N6-((3R)-3,6-diaminohexanoyl)-L-lysyl-[protein] + AMP + diphosphate, RHEA:83435). Yanagisawa et al. showed the protein-lysylation activity with alpha-lysine, and Roy et al. established (R)-beta-lysine as the efficient substrate (about 100-fold better than alpha-lysine or (S)-beta-lysine). The low alpha-lysine activity is not a separate function to annotate. Supporting Evidence: PMID:20729861 it specifically aminoacylates EF-P with lysine PMID:21841797 EF-P was efficiently functionally modified with (R)-Ξ²-lysine but not (S)-Ξ²-lysine or genetically encoded Ξ±-amino acids file:ECOLI/epmA/epmA-uniprot.txt The substrate (R)-beta-lysine is 100-fold more efficient |
| GO:0052868 protein-lysine lysyltransferase activity | IEA GO_REF:0000116 | ACCEPT | Summary: Protein-lysine lysyltransferase activity, from the Rhea mapping of the curated EpmA reaction. Reason: The Rhea reaction is the experimentally characterized EpmA reaction (UniProt cites PMID:21841797 and PMID:22128152), so the IEA agrees with the IDA row. Supporting Evidence: file:ECOLI/epmA/epmA-uniprot.txt Reaction=D-beta-lysine + L-lysyl-[protein] + ATP PMID:21841797 EF-P was efficiently functionally modified with (R)-Ξ²-lysine but not (S)-Ξ²-lysine or genetically encoded Ξ±-amino acids |
| GO:0071468 cellular response to acidic pH | IMP PMID:27826291 Identification of Genes Required for Growth of Escherichia c... | KEEP AS NON CORE | Summary: An epmA deletion grows poorly at pH 4.5, a defect confirmed by in-frame deletion and complementation and linked to reduced CadA (lysine decarboxylase) production. Reason: The phenotype is real and the acts_upstream_of_or_within qualifier is apt, but it is indirect. EpmA activates EF-P, which is needed to translate the polyproline-containing acid-stress regulator CadC (PMID:23239623); the cadBA response then follows. EpmA does none of the work of the acid response itself, so it is kept as a non-core downstream consequence of EF-P modification. Supporting Evidence: PMID:27826291 For mnmE, apaH, epmA, and waaC, de novo in frame deletion and genetic complementation confirmed their role in low pH growth PMID:27826291 encoding the transcriptional regulator LeuO and the elongation factor P-Ξ²-lysine ligase EpmA, can be linked to CadA production PMID:23239623 we demonstrate the physiological relevance of EF-P to fine-tune the expression of the polyproline-containing pH receptor CadC to levels necessary for an appropriate stress response |
Loading supporting contentβ¦
Download this section (compressed HTML)Q: Should PAINT record a loss of tRNA binding, lysine-tRNA ligase activity and lysyl-tRNA aminoacylation at the EpmA subfamily node of PTHR42918, so that these LysRS functions stop propagating to EpmA orthologs?
Q: Should the InterPro2GO mappings of IPR004525 (EpmA) to lysine-tRNA ligase activity and lysyl-tRNA aminoacylation be withdrawn, given that the family is defined by EF-P beta-lysylation?
Q: Does GO need a process term for EF-P lysine beta-lysylation (analogous to peptidyl-hypusine biosynthesis for eIF5A), to replace the general post-translational protein modification term for EpmA and EpmC?
Experiment: Solve EpmA structures with (R)- and (S)-beta-lysine adenylate analogs and test pocket mutants for altered enantiomer and alpha/beta selectivity by ATP-PPi exchange and EF-P modification assays.
Hypothesis: The residues that select (R)-beta-lysine over L-alpha-lysine lie in the amino-acid pocket that EpmA shares with LysRS.
Loading supporting contentβ¦
Download this section (compressed HTML)Loading supporting contentβ¦
Download this section (compressed HTML)