PflA is the pyruvate formate-lyase activating enzyme (PFL-AE, PFL activase; EC 1.97.1.4) of Escherichia coli, the founding member of the radical SAM activases that install glycyl radicals on glycyl radical enzymes. It is a monomeric cytosolic protein of about 28 kDa with a single [4Fe-4S] cluster bound by three cysteines of a CxxxCxxC motif. S-adenosyl-L-methionine (SAM) chelates the fourth, unique iron, and a monovalent cation (K+ gives maximal activity) is bound beside the SAM carboxylate. Reduced flavodoxin reduces the cluster to the active [4Fe-4S]+ state, which cleaves SAM to methionine and a 5'-deoxyadenosyl radical. That radical stereospecifically abstracts the pro-S hydrogen of Gly734 of pyruvate formate-lyase (PflB), converting the inactive enzyme into its active glycyl-radical form. Crystal structures with a Gly734-loop peptide show how the activase reaches the buried glycine. This post-translational activation switches on pyruvate formate-lyase for anaerobic glucose fermentation, and the oxygen-labile cluster interconverts in cells according to oxygen status.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: Radical SAM domain InterPro2GO mapping to the root catalytic activity term. Reason: Per the RADICAL_SAM treatment rule, the root term is replaced by the specific reaction. PflA's reaction is GO:0043365 [formate-C-acetyltransferase]-activating enzyme activity (EC 1.97.1.4), which it carries by IDA and IBA. Proposed replacements: [formate-C-acetyltransferase]-activating enzyme activity Supporting Evidence: PMID:18852451 Pyruvate formate-lyase activating enzyme generates a stable and catalytically essential glycyl radical on G(734) of pyruvate formate-lyase via the direct, stereospecific abstraction of a hydrogen atom from pyruvate formate-lyase. |
| GO:0005515 protein binding | IPI PMID:24016774 Flavodoxin cofactor binding induces structural changes that ... | REMOVE | Summary: Surface plasmon resonance shows that holo-flavodoxin (FldA, P61949) binds PFL-AE, and apo-flavodoxin does not. Reason: The interaction is the physiological electron-donor encounter. Reduced flavodoxin is a cosubstrate in the defining reaction of GO:0043365, so the specific activity term already captures it, and protein binding adds nothing. No more specific binding MF fits an electron-donor encounter of this kind. Supporting Evidence: PMID:24016774 Holo-Fld is capable of associating with NADP(+)-dependent flavodoxin oxidoreductase (FNR) and pyruvate formate-lyase activating enzyme (PFL-AE) whereas there is no detectable interaction between apo-Fld and either protein. |
| GO:0005515 protein binding | IPI PMID:24561554 The binary protein-protein interaction landscape of Escheric... | REMOVE | Summary: Yeast two-hybrid interaction of PflA with the uncharacterised protein YdcY (P64455). Reason: This is an uninformative protein binding row from a binary interactome screen, with no functional context for YdcY. Protein binding is not used as a functional annotation. |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | MODIFY | Summary: UniProt subcellular location mapping to cytoplasm. Reason: The term is correct but general. PflA is a soluble cytosolic enzyme, and the gene already carries the more specific part_of descendant GO:0005829 cytosol by IDA (PMID:15911532) and IBA. Proposed replacements: cytosol Supporting Evidence: file:ECOLI/pflA/pflA-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm. |
| GO:0005829 cytosol | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of cytosolic activity at the PflA activase node. Reason: PflA acts on the soluble PflB dimer in the cytosol, and the IDA fractionation result (PMID:15911532) agrees. PflA appears among the sources of this IBA because it has its own experimental annotation, which marks experimental grounding on the target. Supporting Evidence: PMID:15911532 2,160 were annotated and assigned to the cytosol, periplasm, inner membrane, and outer membrane by biochemical fractionation |
| GO:0005829 cytosol | IDA PMID:15911532 Localization, annotation, and comparison of the Escherichia ... | ACCEPT | Summary: Detected in the cytosolic fraction in a fractionation proteomics survey of E. coli K-12. Reason: The cached paper is abstract-only, but the assignment comes from biochemical fractionation and fits a soluble enzyme acting on cytosolic PFL. It is accepted, deferring to the curator. Supporting Evidence: PMID:15911532 2,160 were annotated and assigned to the cytosol, periplasm, inner membrane, and outer membrane by biochemical fractionation |
| GO:0006974 DNA damage response | IEP PMID:11967071 Over 1000 genes are involved in the DNA damage response of E... | MARK AS OVER ANNOTATED | Summary: pflA transcript level changed after mitomycin C treatment in a genome-wide microarray study in which about 30% of genes changed expression. Reason: A transcriptional change in a global stress profile does not show that PflA does any of the work of the DNA damage response. The study reports expression changes in nearly 30% of all genes, and PflA has no known activity on DNA or in repair or signalling. Its only established function is PFL activation. The row reflects expression, not participation. Supporting Evidence: PMID:11967071 Unexpectedly, a large number of genes (nearly 30% of all genes) displayed significant changes in their expression level. |
| GO:0016491 oxidoreductase activity | IDA PMID:19711960 The iron-sulfur cluster of pyruvate formate-lyase activating... | MODIFY | Summary: Whole-cell Mossbauer study of PFL-AE cluster interconversion, used to support oxidoreductase activity. Reason: The term is correct but general. The activase reaction GO:0043365 (EC 1.97.1.4, an oxidoreductase) is an is_a descendant of GO:0016491, and the gene carries it by IDA. Proposed replacements: [formate-C-acetyltransferase]-activating enzyme activity Supporting Evidence: PMID:19711960 Pyruvate formate-lyase activating enzyme (PFL-AE) catalyzes the generation of a catalytically essential glycyl radical on pyruvate formate-lyase (PFL). |
| GO:0016491 oxidoreductase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping to oxidoreductase activity. Reason: The term is correct but general and is refined to GO:0043365, as for the IDA row. Proposed replacements: [formate-C-acetyltransferase]-activating enzyme activity Supporting Evidence: PMID:19711960 Pyruvate formate-lyase activating enzyme (PFL-AE) catalyzes the generation of a catalytically essential glycyl radical on pyruvate formate-lyase (PFL). |
| GO:0018201 peptidyl-glycine modification | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference that the PflA activase clade modifies a peptidyl-glycine. Reason: PflA converts Gly734 of PflB into a glycyl radical. GO has no glycyl radical formation term (QuickGO search, 2026-10-19), and GO:0018201 is the most specific process that fits. PflA is the enzyme that performs the modification, so it does the work of the process. PflA among its own sources reflects its IDA row. Supporting Evidence: PMID:8175649 Our results show that the radical in pyruvate formatelyase is produced by stereospecific abstraction of the pro-S hydrogen of glycine 734 by the 5'-dAdo radical generated in the active center of PFL activase. |
| GO:0018201 peptidyl-glycine modification | IDA PMID:6364987 Pyruvate formate-lyase (inactive form) and pyruvate formate-... | ACCEPT | Summary: Purified PFL activase converts inactive PFL (Ei) into active PFL (Ea) post-translationally, with SAM cleavage. Reason: This is direct biochemical evidence that PflA performs the post-translational modification of PFL. Later work places the modification on Gly734 (PMID:1310545). The action matches the IBA row. Supporting Evidence: PMID:6364987 The catalytically active form (Ea) of pyruvate formate-lyase in Escherichia coli cells is generated from an inactive form of the enzyme (Ei) through a post-translational process that requires a distinct activating enzyme and is linked to the cleavage of adenosylmethionine to methionine and 5'-deoxyadenosine. PMID:1310545 The N-terminal block on the small fragment was identified by mass spectrometry as an oxalyl residue that derives from Gly-734, thus assigning the primary structural glycyl radical position. |
| GO:0030955 potassium ion binding | IDA PMID:28768413 Monovalent Cation Activation of the Radical SAM Enzyme Pyruv... | KEEP AS NON CORE | Summary: PFL-AE binds a catalytically essential monovalent cation at its active site, and activity is maximal with K+ (NH4+ nearly as good). Reason: The monovalent cation site is real and needed for activity. A SAM carboxylate oxygen ligates the cation, which tunes the SAM-cluster interaction. It is an activating cofactor site, not a separate function, so it is kept as non-core. The crystal structures contain Na+, but K+ is the activating ion. Potassium ion binding is as precise as the data allow. Supporting Evidence: PMID:28768413 We show that PFL-AE binds a catalytically essential monovalent cation at its active site PMID:28768413 PFL-AE activity sharply maximizes at K+, with NH4+ closely matching the efficacy of K+. |
| GO:0043364 glycyl-radical enzyme activating activity | IEA GO_REF:0000116 | MODIFY | Summary: Rhea mapping of the PFL activase reaction to the generic glycyl-radical activase term. Reason: The term is correct but general. RHEA:19225 is the [formate C-acetyltransferase] reaction, which corresponds to the child term GO:0043365. PflA carries that term by IDA and IBA. Proposed replacements: [formate-C-acetyltransferase]-activating enzyme activity Supporting Evidence: file:ECOLI/pflA/pflA-uniprot.txt Reaction=glycyl-[formate C-acetyltransferase] + reduced [flavodoxin] + S-adenosyl-L-methionine = glycin-2-yl radical-[formate C-acetyltransferase] + semiquinone [flavodoxin] + 5'-deoxyadenosine + L-methionine + H(+) |
| GO:0043365 [formate-C-acetyltransferase]-activating enzyme activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference of PFL activase activity at the PflA node. Reason: PflA is the experimentally characterised member of this node and has the activity by direct assay (PMID:6364987, PMID:8175649). Supporting Evidence: PMID:8175649 Our results show that the radical in pyruvate formatelyase is produced by stereospecific abstraction of the pro-S hydrogen of glycine 734 by the 5'-dAdo radical generated in the active center of PFL activase. |
| GO:0043365 [formate-C-acetyltransferase]-activating enzyme activity | IDA PMID:6364987 Pyruvate formate-lyase (inactive form) and pyruvate formate-... | ACCEPT | Summary: The purified activating enzyme converts inactive PFL into the active form, coupled to SAM cleavage to methionine and 5'-deoxyadenosine. Reason: This is the defining activity of PflA (EC 1.97.1.4). It is confirmed by stereospecific Gly734 hydrogen abstraction with dihydroflavodoxin as cosubstrate (PMID:8175649) and by the peptide-bound crystal structure (PMID:18852451). Supporting Evidence: PMID:6364987 The catalytically active form (Ea) of pyruvate formate-lyase in Escherichia coli cells is generated from an inactive form of the enzyme (Ei) through a post-translational process that requires a distinct activating enzyme and is linked to the cleavage of adenosylmethionine to methionine and 5'-deoxyadenosine. PMID:8175649 post-translationally by pyruvate formate-lyase activating enzyme (PFL activase) using S-adenosylmethionine (AdoMet) and dihydroflavodoxin as co-substrates |
| GO:0043365 [formate-C-acetyltransferase]-activating enzyme activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined automated annotation from the PFL activase InterPro family and EC 1.97.1.4. Reason: Agrees with the IDA evidence. Supporting Evidence: PMID:18852451 Pyruvate formate-lyase activating enzyme generates a stable and catalytically essential glycyl radical on G(734) of pyruvate formate-lyase via the direct, stereospecific abstraction of a hydrogen atom from pyruvate formate-lyase. |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000002 | MODIFY | Summary: Radical SAM domain InterPro2GO mapping to iron-sulfur cluster binding. Reason: Per the RADICAL_SAM treatment rule, refined to GO:0051539, which the gene carries by IDA (PMID:9478932). Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: PMID:9478932 The previously inferred metallic cofactor was identified as redox-interconvertible polynuclear iron-sulfur cluster, most probably of the [4Fe-4S] type, according to UV-visible and EPR spectroscopic information. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IDA PMID:9478932 Reconstitution and characterization of the polynuclear iron-... | ACCEPT | Summary: Reconstituted PFL activase contains a redox-interconvertible [4Fe-4S] cluster, and Cys29, Cys33 and Cys36 are essential for active holoenzyme. Reason: The radical SAM cluster is the catalytic cofactor. Its [4Fe-4S] nature is confirmed in vitro and in whole cells (PMID:19711960), and the crystal structure shows the cluster bound to SAM (PMID:18852451). Supporting Evidence: PMID:9478932 Cys --> Ser replacements by site-directed mutagenesis determined Cys-29, Cys-33, and Cys-36 to be essential to yield active holoenzyme. PMID:19711960 Purified PFL-AE contains an oxygen-sensitive, labile [4Fe-4S] cluster that undergoes cluster interconversions in vitro, with only the [4Fe-4S](+) cluster state being catalytically active. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping to [4Fe-4S] cluster binding. Reason: Agrees with the IDA evidence and the RADICAL_SAM treatment rule. Supporting Evidence: PMID:9478932 Cys --> Ser replacements by site-directed mutagenesis determined Cys-29, Cys-33, and Cys-36 to be essential to yield active holoenzyme. |
| GO:0051604 protein maturation | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT inference that the PflA clade acts in protein maturation. Reason: Installing the Gly734 radical is how PFL attains its full functional capacity, which matches the definition of GO:0051604. PflA catalyses this step. No more specific child of protein maturation describes radical installation, so the term is as precise as GO allows. Supporting Evidence: PMID:6364987 The catalytically active form (Ea) of pyruvate formate-lyase in Escherichia coli cells is generated from an inactive form of the enzyme (Ei) through a post-translational process that requires a distinct activating enzyme and is linked to the cleavage of adenosylmethionine to methionine and 5'-deoxyadenosine. |
| GO:0051604 protein maturation | IDA PMID:6364987 Pyruvate formate-lyase (inactive form) and pyruvate formate-... | ACCEPT | Summary: The activating enzyme converts inactive PFL into active PFL. Reason: This is direct evidence that PflA catalyses the maturation step. The action matches the IBA row. Supporting Evidence: PMID:6364987 The catalytically active form (Ea) of pyruvate formate-lyase in Escherichia coli cells is generated from an inactive form of the enzyme (Ei) through a post-translational process that requires a distinct activating enzyme and is linked to the cleavage of adenosylmethionine to methionine and 5'-deoxyadenosine. |
| GO:1904047 S-adenosyl-L-methionine binding | IDA PMID:9478932 Reconstitution and characterization of the polynuclear iron-... | NEW | Summary: SAM binds the cluster-containing holoenzyme and shifts the EPR signal of the reduced [4Fe-4S]+ cluster. The crystal structure shows SAM chelating the unique iron. Reason: GOA has no SAM-binding row for PflA. Following the RADICAL_SAM Phase 1 convention, GO:1904047 is added for every member whose radical SAM catalysis is established. SAM binding is shown spectroscopically (PMID:9478932) and structurally (PMID:18852451, with experimentally annotated SAM-binding residues in UniProt). This is a molecular function of PflA itself. Supporting Evidence: PMID:9478932 The iron-sulfur cluster complement proved to be a prerequisite for effective binding of adenosylmethionine, which induces a characteristic shift of the EPR signal shape of the reduced enzyme form ([4Fe-4S]+) from axial to rhombic symmetry. PMID:24016774 The SAM cofactor coordinates the unique iron of the [4Fe-4S] cluster via amino and carboxylate moieties |
Loading supporting content…
Download this section (compressed HTML)Q: Does the K+ dependence of PFL-AE make PFL activation sensitive to intracellular potassium or ammonium levels under physiological anaerobic conditions?
Q: How does PFL-AE gain access to Gly734, which is buried in the PFL dimer, and is a large conformational change of the PflB glycyl radical loop required in vivo?
Experiment: Compare the PFL glycyl radical content (whole-cell EPR) of anaerobic cells expressing wild-type PFL-AE or a cation-site variant across a range of external K+ concentrations in a K+-uptake-deficient background.
Hypothesis: Intracellular K+ modulates PFL activation in vivo through the PFL-AE monovalent cation site.
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)