pqqE (PP_0376) encodes the radical-SAM/RS-SPASM PqqA peptide cyclase that initiates pyrroloquinoline quinone (PQQ) biosynthesis by catalyzing the first committed step, formation of an intramolecular carbon-carbon cross-link between a conserved glutamate and a tyrosine residue in the ribosomally synthesized PqqA precursor peptide (EC 1.21.98.4). The enzyme uses a SAM-cleaving [4Fe-4S] radical-SAM cluster plus auxiliary SPASM-domain Fe-S clusters, and productive turnover requires the peptide chaperone PqqD, which presents PqqA to PqqE as a ternary PqqA-PqqD-PqqE complex. Its core function is cytosolic PqqA peptide cyclization with required SAM and Fe-S cofactors.
Definition: Catalysis of radical-SAM-dependent formation of an intramolecular carbon-carbon cross-link between glutamate and tyrosine residues in the PqqA precursor peptide during pyrroloquinoline quinone biosynthesis.
Justification: GO:0009975 cyclase activity is the best available current molecular-function term, but PqqE catalyzes a specific PqqA peptide cross-linking reaction that is not captured by a dedicated GO term. The proposed activity corresponds to EC 1.21.98.4 and is supported by the PqqE/PqqD radical-SAM biochemical evidence in PMID:26961875 and by the deep research synthesis.
Parent term: cyclase activity
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: This very broad catalytic activity annotation is true but not useful for PqqE because the record already has the more informative cyclase activity and pathway annotations. Reason: Following the RADICAL_SAM project rule for the generic domain-derived rows (projects/RADICAL_SAM.md): the row is true, so it is refined rather than flagged as an over-annotation. GO:0009975 cyclase activity, already carried by this entry, is the reaction term. Proposed replacements: cyclase activity Supporting Evidence: file:PSEPK/pqqE/pqqE-uniprot.txt RecName: Full=PqqA peptide cyclase |
| GO:0005506 iron ion binding | IEA GO_REF:0000104 | MODIFY | Summary: Iron ion binding is directionally correct for a radical-SAM [4Fe-4S] enzyme, but it is less specific than the 4 iron, 4 sulfur cluster binding annotation. Deep research shows PqqE binds multiple Fe-S clusters (the SAM-cleaving RS cluster plus auxiliary SPASM clusters), so the iron-sulfur cluster terms capture the cofactor more precisely than bare iron ion binding. Reason: Following the RADICAL_SAM project rule for the generic domain-derived rows (projects/RADICAL_SAM.md): a lateral correction, because the record assigns the iron to a [4Fe-4S] cluster and records no mononuclear iron site. Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: file:PSEPK/pqqE/pqqE-uniprot.txt Name=[4Fe-4S] cluster; Xref=ChEBI:CHEBI:49883; |
| GO:0009975 cyclase activity | IEA GO_REF:0000104 | ACCEPT | Summary: Cyclase activity is the best available current molecular-function term for PqqE. UniProt names the protein PqqA peptide cyclase and describes cross-link formation in PqqA. Deep research independently confirms PqqE is the initiating radical-SAM/SPASM peptide cyclase that forms the first intramolecular C-C cross-link in the PqqA precursor between a conserved glutamate and tyrosine. Reason: A more specific PqqA peptide cyclase term would be preferable (see proposed_new_terms), but GO:0009975 captures the core catalytic category and is strongly supported by both primary biochemical work and the deep research synthesis. Supporting Evidence: file:PSEPK/pqqE/pqqE-uniprot.txt RecName: Full=PqqA peptide cyclase file:PSEPK/pqqE/pqqE-uniprot.txt Catalyzes the cross-linking of a glutamate residue and a PMID:26961875 PqqE, in conjunction with PqqD, carries out the first step in PQQ biosynthesis file:PSEPK/pqqE/pqqE-deep-research-falcon.md PqqE is the initiating enzyme file:PSEPK/pqqE/pqqE-deep-research-falcon.md formation of a new intramolecular carbon–carbon bond within PqqA between a conserved glutamate and tyrosine |
| GO:0018189 pyrroloquinoline quinone biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: This process annotation is correct. PqqE catalyzes the first committed PqqA peptide cross-linking step in pyrroloquinoline quinone biosynthesis. Deep research confirms PqqE is one component of the conserved pqqA-E operon and acts as the pathway's initiating radical-SAM cross-linking enzyme. Reason: PqqE is a core enzyme of the PQQ pathway, catalyzing the initiating committed step. Supporting Evidence: file:PSEPK/pqqE/pqqE-uniprot.txt tyrosine residue in the PqqA protein as part of the biosynthesis of PMID:26961875 PqqE, in conjunction with PqqD, carries out the first step in PQQ biosynthesis file:PSEPK/pqqE/pqqE-deep-research-falcon.md catalyzing the first committed chemical transformation |
| GO:0046872 metal ion binding | IEA GO_REF:0000002 | MODIFY | Summary: Metal ion binding is too generic for PqqE. The biologically meaningful metal cofactor is the [4Fe-4S] radical-SAM cluster (plus auxiliary SPASM Fe-S clusters). Reason: Following the RADICAL_SAM project rule for the generic domain-derived rows (projects/RADICAL_SAM.md): the same lateral correction, and never MARK_AS_OVER_ANNOTATED: PqqE does bind metal, as clusters. Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: file:PSEPK/pqqE/pqqE-uniprot.txt Name=[4Fe-4S] cluster; Xref=ChEBI:CHEBI:49883; |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000002 | MODIFY | Summary: Iron-sulfur cluster binding is correct but less specific than the 4 iron, 4 sulfur cluster binding annotation that matches the radical-SAM cofactor. Deep research describes the RS [4Fe-4S] cluster plus auxiliary SPASM clusters (an unusual AuxI [2Fe-2S] and an AuxII [4Fe-4S]), so the generic iron-sulfur term remains a useful parent covering all of these. Reason: Following the RADICAL_SAM project rule for the generic domain-derived rows (projects/RADICAL_SAM.md): correct but general. The earlier reason cited an auxiliary [2Fe-2S] cluster, but this entry records no [2Fe-2S] cofactor and the cited analysis gives AuxII as [4Fe-4S], so GO:0051539 covers every cluster recorded for the protein and entails this parent. Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: file:PSEPK/pqqE/pqqE-uniprot.txt Name=[4Fe-4S] cluster; Xref=ChEBI:CHEBI:49883; file:PSEPK/pqqE/pqqE-deep-research-falcon.md AuxII:** a **[4Fe–4S]** cluster ligated by **3 Cys + 1 Asp |
| GO:0051539 4 iron, 4 sulfur cluster binding | IEA GO_REF:0000002 | ACCEPT | Summary: This is the appropriate cofactor-binding annotation for PqqE. The protein is a radical-SAM enzyme with a SAM-cleaving [4Fe-4S] cluster (and an additional auxiliary [4Fe-4S] cluster in the SPASM domain). Deep research confirms the RS [4Fe-4S] cluster reductively cleaves SAM to generate the 5'-deoxyadenosyl radical that initiates catalysis. Reason: The [4Fe-4S] cluster is mechanistically central to radical-SAM peptide cross-linking. Supporting Evidence: file:PSEPK/pqqE/pqqE-uniprot.txt [4Fe-4S] cluster file:PSEPK/pqqE/pqqE-deep-research-falcon.md One-electron reduction of the RS [4Fe–4S] cluster enables SAM cleavage to methionine + 5′-dAdo• |
| GO:1904047 S-adenosyl-L-methionine binding | IEA GO_REF:0000104 | ACCEPT | Summary: S-adenosyl-L-methionine binding is appropriate for PqqE because radical-SAM chemistry uses SAM to generate the radical species required for peptide cross-linking. Deep research confirms the RS [4Fe-4S] cluster reductively cleaves SAM to methionine plus the 5'-deoxyadenosyl radical that abstracts a hydrogen from the PqqA glutamate side chain. Reason: SAM binding is a mechanistically relevant cofactor interaction for the core enzyme activity. Supporting Evidence: file:PSEPK/pqqE/pqqE-uniprot.txt S-adenosyl-L-methionine file:PSEPK/pqqE/pqqE-deep-research-falcon.md it uses a **[4Fe–4S] cluster** to reductively cleave **S-adenosyl-L-methionine (SAM)** to generate a **5′-deoxyadenosyl radical |
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Download this section (compressed HTML)Q: Which PqqE radical-SAM intermediates can be observed during PqqA Glu-Tyr cross-link formation in KT2440?
Experiment: Purify PqqE with reconstituted [4Fe-4S] cluster and assay SAM-dependent cross-linking of synthetic or recombinant PqqA peptide by LC-MS/MS, with and without PqqD, using a physiological flavodoxin/FNR/NADPH electron-delivery system.
Hypothesis: PqqE initiates PQQ biosynthesis by radical-SAM cross-linking of PqqA, and this requires the PqqD chaperone.
Type: in vitro radical-SAM enzyme assay
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