nirJ encodes a radical S-adenosyl-L-methionine (SAM) enzyme of the heme d1 biosynthesis pathway, encoded in the nirSMCFDLGHJEN cluster that supports anaerobic denitrification through cytochrome cd1 nitrite reductase (NirS). Heme d1, the iron dioxo-isobacteriochlorin cofactor of NirS, is made from siroheme; NirJ acts on the intermediate 12,18-didecarboxysiroheme, cleaving off the propionate side chains of rings A and B with release of acrylate. The protein has an N-terminal radical SAM domain with the CxxxCxxC motif that binds the SAM-cleaving [4Fe-4S] cluster and a C-terminal SPASM-type region carrying an auxiliary [4Fe-4S] cluster. Insertional inactivation of nirJ abolishes heme d1 formation in P. aeruginosa.
Definition: Catalysis of the S-adenosyl-L-methionine-dependent radical removal of the propionate side chains from pyrrole rings A and B of 12,18-didecarboxysiroheme, with release of acrylate, during heme d1 biosynthesis.
Justification: The NirJ reaction has been shown in vitro but has no GO molecular function term, so NirJ proteins can only be annotated to catalytic activity.
Parent term: catalytic activity
Supporting Evidence:
Definition: The chemical reactions and pathways resulting in the formation of heme d1, the iron dioxo-isobacteriochlorin prosthetic group of cytochrome cd1 nitrite reductase, from siroheme.
Justification: Heme d1 enzymes (NirF, NirDLGH, NirJ, NirE) can only be annotated to the broad heme biosynthetic process, which most readers take to mean protoheme; a child term parallel to siroheme biosynthetic process would make these annotations precise.
Parent term: heme biosynthetic process
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO root-level catalytic activity from radical SAM signatures. Reason: True but uninformative. The activity of NirJ is known from ortholog biochemistry (SAM-dependent removal of two propionate side chains from 12,18-didecarboxysiroheme), but GO has no term for it, so the replacement is a proposed new term. Proposed replacements: 12,18-didecarboxysiroheme propionate-cleaving radical SAM activity Supporting Evidence: PMID:29076625 we show that NirJ indeed catalyzes the removal of the two propionate side chains under simultaneous SAM cleavage. |
| GO:0006783 heme biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA from PTN000132400, whose sole seed is this gene's PseudoCAP IMP (PMID:8982003). Correct for NirJ in GO's broad sense of heme (heme d1); the node is an archaeal SPASM radical SAM clade that the term does not suit. Reason: For NirJ the term is right and the most precise available (heme d1; GO places siroheme biosynthesis under GO:0006783, so iron isobacteriochlorins count as heme). The target's own experimental row is the seed. The defect is at the node, whose other leaves are mostly archaeal SPASM radical SAM proteins of unrelated or unknown function; recorded as a GO_CENTRAL defect. Propagation Review Root cause: PROPAGATION BAD Failure modes: WRONG ORTHOLOG OR PARALOG Sources checked: PANTHER:PTN000132400 · PTN000132400 SUPPORTS SOURCE BUT NOT TARGET QuickGO (2026-10-28) lists 15 GO:0006783 IBA leaves in 6 taxa. Apart from NirJ they are archaeal (Korarchaeum, Methanosarcina, Pyrobaculum, Sulfolobus, Halobacterium) SPASM radical SAM proteins; only M. acetivorans Q8TLK1 (TIGR04546, AhbC) is a heme-pathway enzyme. UniProtKB:G3XD10 · nirJ SUPPORTS TRANSFER The target's own experimental annotation is the IBD seed; expected, not circular. Supporting Evidence: PMID:8982003 Insertional mutation and complementation analyses showed that all of the nirFDLGHJE genes were necessary for the biosynthesis of heme d1. PMID:40318205 the removal of two propionate side chains, the latter being catalyzed by the radical SAM enzyme NirJ |
| GO:0006783 heme biosynthetic process | IMP PMID:8982003 Gene cluster for dissimilatory nitrite reductase (nir) from ... | ACCEPT | Summary: PseudoCAP IMP; insertion and complementation show nirJ is needed for heme d1 synthesis. Sole seed of the GO:0006783 IBD at PTN000132400. Reason: The paper's mutant result shows necessity, and complementation addresses polarity on the downstream nirE. Participation (not just necessity) is established by later biochemistry showing that NirJ itself removes the two propionate side chains from 12,18-didecarboxysiroheme. GO has no heme d1 term, and siroheme biosynthesis is already a child of GO:0006783, so this is the most precise term available. A heme d1 biosynthetic process NTR is proposed. Supporting Evidence: PMID:8982003 Insertional mutation and complementation analyses showed that all of the nirFDLGHJE genes were necessary for the biosynthesis of heme d1. PMID:29076625 we show that NirJ indeed catalyzes the removal of the two propionate side chains under simultaneous SAM cleavage. |
| GO:0046872 metal ion binding | IEA GO_REF:0000104 | MODIFY | Summary: UniRule-derived generic metal binding. Reason: Correct but general; the metal is iron in [4Fe-4S] clusters, already captured as GO:0051539. Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: PMID:20420837 The purified protein was found to contain a 4Fe-4S centre, in line with the prediction that it belongs to the radical SAM class of enzymes. |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO generic Fe-S binding from radical SAM signatures. Reason: Correct but general; both clusters of NirJ are [4Fe-4S] (GO:0051539, already carried). Proposed replacements: 4 iron, 4 sulfur cluster binding Supporting Evidence: PMID:40318205 Here, we demonstrate by Mössbauer spectroscopy that NirJ contains a [4Fe-4S] cluster ligated by cysteine residues as its auxiliary cluster. |
| GO:0051539 4 iron, 4 sulfur cluster binding | IEA GO_REF:0000120 | ACCEPT | Summary: Combined IEA from the NirJ family signature and UniRule. Reason: PA0511 has the radical SAM CxxxCxxC cluster-binding cysteines, and NirJ orthologs carry a SAM-binding [4Fe-4S] cluster plus an auxiliary [4Fe-4S] cluster, as required for catalysis. Supporting Evidence: PMID:29076625 Here, we demonstrate that NirJ is a Radical SAM enzyme carrying two iron-sulfur clusters. PMID:40318205 Here, we demonstrate by Mössbauer spectroscopy that NirJ contains a [4Fe-4S] cluster ligated by cysteine residues as its auxiliary cluster. |
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Download this section (compressed HTML)Q: Does P. aeruginosa NirJ itself also introduce the keto groups at C3 and C8, or is a second enzyme (for example NirN or NirF) required?
Experiment: Purify P. aeruginosa NirJ (PA0511) and assay propionate removal from 12,18-didecarboxysiroheme, alone and with NirN/NirF, to identify the product and whether the keto groups are formed.
Type: in vitro enzyme assay with HPLC-MS product analysis
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