HemN (also called CgdH) is the oxygen-independent coproporphyrinogen III dehydrogenase of Escherichia coli, a cytoplasmic, monomeric radical S-adenosyl-L-methionine (SAM) enzyme that converts coproporphyrinogen III to protoporphyrinogen IX in the late, shared part of heme biosynthesis. It oxidatively decarboxylates the propionate side chains of pyrrole rings A and B to vinyl groups without using molecular oxygen, and so provides this step under anaerobic conditions, where the oxygen-dependent isoenzyme HemF cannot act. A [4Fe-4S] cluster ligated by the three cysteines of the CX3CX2C motif binds one SAM at its unique iron; reductive cleavage of that SAM yields a 5'-deoxyadenosyl radical, and a second SAM bound alongside acts as a radical relay, so that two SAM are consumed for each protoporphyrinogen IX formed. Catalysis proceeds through stereospecific abstraction of the pro-S hydrogen at the beta carbon of a propionate side chain, giving an allylic substrate radical that then loses CO2. The 2.07 A crystal structure of E. coli HemN was the first structure of any radical SAM enzyme. HemN is the founding member of a family whose sister subfamily, HemW, lacks this activity and instead acts as a heme chaperone.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: Root-level catalytic activity from the radical SAM InterPro signatures. HemN's reaction is known and directly demonstrated: anaerobically purified recombinant enzyme converts coproporphyrinogen III to protoporphyrinogen IX with SAM as cosubstrate (PMID:12114526; PMID:15967800). Reason: Per the RADICAL_SAM project's treatment rule for generic rows, GO:0003824 is replaced by the specific reaction term the gene already carries by IDA, IBA and IEA: GO:0051989 coproporphyrinogen dehydrogenase activity. Proposed replacements: coproporphyrinogen dehydrogenase activity Supporting Evidence: PMID:12114526 A newly established anaerobic enzyme assay was used to demonstrate for the first time in vitro coproporphyrinogen-III oxidase activity for recombinant purified HemN file:ECOLI/hemN/hemN-uniprot.txt Reaction=coproporphyrinogen III + 2 S-adenosyl-L-methionine = |
| GO:0004109 coproporphyrinogen oxidase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from IPR004558 (Coprogen_oxidase_HemN). In current GO, GO:0004109 is defined by the oxygen-dependent reaction (coproporphyrinogen III + 2 H+ + O2 = 2 CO2 + 2 H2O + protoporphyrinogen IX), which is the HemF-type reaction. HemN performs the same overall conversion but with SAM, not O2, as the oxidant: the reaction GO represents as GO:0051989 coproporphyrinogen dehydrogenase activity (EC 1.3.98.3, RHEA:15425), which UniProt records for this protein. Reason: The essence (the coproporphyrinogen III to protoporphyrinogen IX step) is right, but the term names the wrong chemistry: HemN is an oxygen-independent enzyme, active in anaerobically purified form, that consumes two SAM per turnover. This is a lateral correction to GO:0051989, the term the gene already carries by IDA. The same correction was made for Pseudomonas putida HemN (genes/PSEPK/hemN), and the RSAD1 review noted that the InterPro mapping to GO:0004109 does not match the family's chemistry. The InterPro2GO mapping of IPR004558 to GO:0004109 should be revised to GO:0051989. Proposed replacements: coproporphyrinogen dehydrogenase activity Supporting Evidence: PMID:15967800 Two molecules of AdoMet were cleaved during one catalytic cycle for the formation of one molecule of protoporphyrinogen IX file:ECOLI/hemN/hemN-uniprot.txt Reaction=coproporphyrinogen III + 2 S-adenosyl-L-methionine = PMID:12975365 two structurally unrelated enzymes, one oxygen-dependent (HemF) and one oxygen-independent (HemN) |
| GO:0005737 cytoplasm | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of cytoplasmic location, with E. coli HemN's own IDA among the descendant evidence. HemN is a soluble enzyme that requires additional cytoplasmic components for activity in vitro (PMID:12114526). Reason: Correct and as specific as warranted for a bacterial soluble enzyme; the core location of the coproporphyrinogen dehydrogenase activity. HemN appearing in its own WITH/FROM reflects its experimental grounding, not circularity. Supporting Evidence: file:ECOLI/hemN/hemN-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0005737 cytoplasm | IDA PMID:12114526 Oxygen-independent coproporphyrinogen-III oxidase HemN from ... | ACCEPT | Summary: Layer et al. 2002 purified HemN as a soluble monomeric protein and found that the enzyme needs additional cytoplasmic components for catalysis. The cached record is abstract-only; UniProt cites this paper for the cytoplasmic location. Reason: Consistent with the soluble, cytoplasmic nature of the enzyme and with the IBA and IEA rows for the same term. Core location. Supporting Evidence: PMID:12114526 The enzyme requires S-adenosyl-l-methionine (SAM), NAD(P)H, and additional cytoplasmatic components for catalysis file:ECOLI/hemN/hemN-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm {ECO:0000269|PubMed:12114526}. |
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: Cytoplasm from UniProt's subcellular-location mapping, which rests on the IDA above. Reason: Consistent with the experimental and IBA rows for this term. Supporting Evidence: file:ECOLI/hemN/hemN-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0006779 porphyrin-containing compound biosynthetic process | IBA GO_REF:0000033 | MODIFY | Summary: Phylogenetic inference that HemN takes part in porphyrin biosynthesis. For E. coli HemN this is directly true: it catalyses the coproporphyrinogen III to protoporphyrinogen IX step of heme biosynthesis (PMID:14633981; PMID:15967800). The node PTN000358281 also spans the HemW subfamily, for which this process is wrong (see genes/ECOLI/hemW and genes/human/RSAD1), but HemN sits in the catalytic clade, so the transfer to this target is sound. Reason: Correct but general. In E. coli the protoporphyrinogen IX that HemN makes is used to make protoheme (heme b, and from it hemes o and d); siroheme branches off earlier at uroporphyrinogen III and does not pass through this step. The specific endpoint is GO:0006785 heme B biosynthetic process, an is_a descendant of GO:0006779 via heme biosynthetic process; this matches the refinement made for P. putida HemN (genes/PSEPK/hemN). The term for the step itself, protoporphyrinogen IX biosynthetic process (GO:0006782), is obsolete. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000358281 SUPPORTS TRANSFER For HemN the node's biosynthetic claim is right: the donors are HemN-type coproporphyrinogen dehydrogenases, and E. coli HemN is itself one. A node this deep can only state the general porphyrin-biosynthesis parent, so the row is refined to the heme B endpoint rather than removed. The same node is placed too deep for the HemW subfamily, which descends from it but lacks the activity. Proposed replacements: heme B biosynthetic process Supporting Evidence: PMID:14633981 HemN catalyzes the essential conversion of coproporphyrinogen III to protoporphyrinogen IX during heme biosynthesis file:ECOLI/hemN/hemN-uniprot.txt protoporphyrin-IX biosynthesis; protoporphyrinogen-IX from coproporphyrinogen-III (AdoMet route): step 1/1. |
| GO:0006779 porphyrin-containing compound biosynthetic process | IEA GO_REF:0000002 | MODIFY | Summary: The same process claim mapped from the HemN-specific InterPro signature, which correctly identifies the catalytic clade. Reason: Same judgement as the IBA row for this term: true, and refined to GO:0006785 heme B biosynthetic process. Proposed replacements: heme B biosynthetic process Supporting Evidence: PMID:15967800 catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX during bacterial heme biosynthesis |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000002 | MODIFY | Summary: Generic iron-sulfur cluster binding from the radical SAM signatures. The cluster is a [4Fe-4S] cluster, shown by EPR and Mossbauer spectroscopy and in the crystal structure (PMID:15967800; PMID:14633981). Reason: Per the RADICAL_SAM treatment rule for generic rows, replace with GO:0051539, the descendant the radical SAM cluster always satisfies and which the gene already carries by IDA and IBA. Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: PMID:14633981 HemN binds a 4Fe-4S cluster through three cysteine residues conserved in all Radical SAM enzymes |
| GO:0051539 4 iron, 4 sulfur cluster binding | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of [4Fe-4S] cluster binding across the HemN/HemW family, with E. coli HemN's own experimental annotation among the descendant evidence. Sound node placement: the CX3CX2C cluster motif is conserved in both subfamilies. Reason: The radical SAM [4Fe-4S] cluster is the catalytic cofactor of HemN, so under the RADICAL_SAM treatment rule this is core for an active member. Supporting Evidence: PMID:15967800 Mössbauer spectroscopy of anaerobically purified HemN has identified a predominantly [4Fe-4S](2+) cluster file:ECOLI/hemN/hemN-uniprot.txt Binds 1 [4Fe-4S] cluster. The cluster is coordinated with 3 cysteines and an exchangeable S-adenosyl-L-methionine. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IDA PMID:12114526 Oxygen-independent coproporphyrinogen-III oxidase HemN from ... | ACCEPT | Summary: Layer et al. 2002 detected an oxygen-sensitive iron-sulfur cluster by absorption spectroscopy and iron analysis and showed that Cys62, Cys66 and Cys69 of the CXXXCXXC motif are essential for cluster formation and activity. Its [4Fe-4S] nature was then established by EPR and Mossbauer spectroscopy (PMID:15967800) and in the crystal structure (PMID:14633981). Reason: Experimentally established catalytic cofactor; core per the RADICAL_SAM treatment rule. Supporting Evidence: PMID:12114526 Cysteine residues Cys(62), Cys(66), and Cys(69), which are part of the conserved CXXXCXXC motif found in all HemN proteins, are essential for iron-sulfur cluster formation and enzyme function PMID:14633981 HemN binds a 4Fe-4S cluster through three cysteine residues conserved in all Radical SAM enzymes |
| GO:0051989 coproporphyrinogen dehydrogenase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Phylogenetic inference of coproporphyrinogen dehydrogenase activity from the HemN-clade node PTN000358335, with E. coli HemN's own IDA among the descendant evidence. The PAINT curator placed this activity at a node below the HemN/HemW split, so it is not propagated to HemW, which matches the experimental finding that HemW lacks the activity (PMID:29282292). Reason: Correct node placement and the core molecular function of HemN. The gene appearing in its own WITH/FROM marks experimental grounding on the target itself. Supporting Evidence: PMID:15967800 Two molecules of AdoMet were cleaved during one catalytic cycle for the formation of one molecule of protoporphyrinogen IX |
| GO:0051989 coproporphyrinogen dehydrogenase activity | IDA PMID:12114526 Oxygen-independent coproporphyrinogen-III oxidase HemN from ... | ACCEPT | Summary: First in vitro demonstration of oxygen-independent coproporphyrinogen III conversion by recombinant, anaerobically purified E. coli HemN, which needs SAM and NAD(P)H. Mutating the cluster cysteines or the GGGTP SAM-binding motif (Gly111, Gly113) abolished activity. Later work showed that two SAM are cleaved for each protoporphyrinogen IX formed, as GO:0051989's equation requires (PMID:15967800), and characterized the substrate radical (PMID:16606627). Reason: The defining activity of the protein, directly demonstrated, and the exact GO reaction (EC 1.3.98.3, RHEA:15425). Core. Supporting Evidence: PMID:12114526 A newly established anaerobic enzyme assay was used to demonstrate for the first time in vitro coproporphyrinogen-III oxidase activity for recombinant purified HemN PMID:15967800 identified AdoMet as a co-substrate for HemN catalysis |
| GO:0051989 coproporphyrinogen dehydrogenase activity | IEA GO_REF:0000120 | ACCEPT | Summary: The same activity from UniProt's combined electronic pipeline (EC 1.3.98.3 / RHEA:15425), reinforcing the IDA and IBA rows. Reason: Consistent with the experimental and phylogenetic rows for this term. Supporting Evidence: file:ECOLI/hemN/hemN-uniprot.txt Evidence={ECO:0000269|PubMed:12114526}; |
| GO:1904047 S-adenosyl-L-methionine binding | IDA PMID:14633981 Crystal structure of coproporphyrinogen III oxidase reveals ... | NEW | Summary: HemN binds two SAM molecules. In the crystal structure one SAM coordinates the unique iron of the [4Fe-4S] cluster and a second binds immediately beside it (PMID:14633981). Spectroscopy shows SAM binding changes the unique iron, and mutating the second SAM site abolishes catalysis (PMID:15967800). Reason: GOA has no SAM-binding row for HemN, although the RADICAL_SAM project treats GO:1904047 as a core cosubstrate-binding function of every active radical SAM enzyme. This is a molecular function the protein itself performs, so the participation test is met directly. HemN has the most direct evidence possible: SAM is resolved in its crystal structure (PDB 1OLT). Comparator check, against GOA rather than against this project's own proposals: GOA does annotate GO:1904047 to radical SAM enzymes in exactly this role, by UniRule -- ECOLI/queE (UR000100349), ECOLI/moaA and PSEPK/moaA (UR000101305), METEA/pqqE and PSEPK/pqqE (UR000098303). So the absence here is patchy UniRule coverage, which rules happen to carry the term, not a GO convention against annotating a radical SAM enzyme's own cosubstrate. That is also why those five genes have an existing row to ACCEPT or MODIFY while this one needs a NEW row. Supporting Evidence: PMID:14633981 A juxtaposed SAM coordinates the fourth Fe ion through its amide nitrogen and carboxylate oxygen PMID:14633981 HemN, strikingly, binds a second SAM immediately adjacent to the first PMID:15967800 However, all mutants lost their overall catalytic ability indicating a functional role for AdoMet2 in HemN catalysis |
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Download this section (compressed HTML)Q: What is the physiological electron donor that reduces the HemN [4Fe-4S] cluster in E. coli? In vitro activity needs NAD(P)H and unidentified cytoplasmic components.
Suggested experts: Gunhild Layer, Dieter Jahn
Q: Can the IPR004558 InterPro2GO mapping be changed from GO:0004109 (oxygen-dependent coproporphyrinogen oxidase) to GO:0051989 (coproporphyrinogen dehydrogenase), which is the reaction HemN-family proteins actually carry out?
Experiment: Determine an anaerobic structure of HemN in complex with coproporphyrinogen III, or a stable analogue, and both SAMs, to measure the SAM1 C5' to SAM2 methyl distance (about 7 A in the substrate-free structure).
Hypothesis: With coproporphyrinogen III bound, the second SAM moves close enough to SAM1 for hydrogen abstraction by the 5'-deoxyadenosyl radical.
Type: anaerobic X-ray crystallography / cryo-EM
Experiment: Fractionate E. coli cytoplasm for the activity needed to support HemN turnover, and test purified flavodoxin (FldA) with flavodoxin-NADP+ reductase (Fpr) in place of crude extract.
Hypothesis: A specific flavodoxin/flavodoxin reductase system is the physiological reductant of HemN.
Type: biochemical reconstitution
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