Protoporphyrin-dependent heme B biosynthesis through alternative entry and oxidation routes

A reusable cross-taxon model of protoporphyrin-dependent heme B biosynthesis. C5 glutamyl-tRNA and C4/Shemin reactions can independently supply 5-aminolevulinate (ALA); many organisms encode one route, while some encode both. A shared tetrapyrrole trunk then feeds independently selected oxygen-dependent or oxygen-independent coproporphyrinogen oxidases and HemJ-, HemG-, or HemY/PPOX-family protoporphyrinogen oxidases. All realizations converge at protoporphyrin IX, which a species-neutral ferrochelatase reaction converts to heme B. This protoporphyrin-dependent (PPD, "classical") route is one of three known routes to heme B; the coproporphyrin-dependent (CPD) route of Firmicutes and Actinobacteria and the siroheme-dependent alternative heme biosynthesis (AHB) route of sulfate-reducing bacteria and archaea share the ALA-to- uroporphyrinogen III core but are outside this module.

Scope is CONCRETE because this document represents a chemically defined, leaf-grounded heme B biosynthetic pathway rather than a gene-free abstract motif; cross-taxon reuse does not make the pathway abstract. The boundary begins with route-specific ALA formation and ends with heme B. HemA/HemL and the trunk through uroporphyrinogen III can also supply sibling tetrapyrrole pathways, but siroheme-, corrin-, and cobalamin-specific enzymes remain outside. GltX supplies glutamyl-tRNA used by both translation and HemA and is upstream of the committed C5 entry chemistry. Coproheme synthesis, heme uptake, storage, degradation, and conversion of heme B to modified hemes are outside the boundary. The module covers only the protoporphyrin-dependent (PPD) route, although GO:0006785 also covers heme B made by the coproporphyrin-dependent (CPD) route (CgoX/HemY, CpfC/HemH and ChdC/HemQ, with AhbD as an oxygen-independent coproheme decarboxylase in some taxa) and by the siroheme-dependent AHB route (AhbA/B, AhbC, AhbD) (PMID:42714178, PMID:25646457, PMID:21969545). CPD and AHB are not modeled here as variants: they leave the shared trunk at coproporphyrinogen III or uroporphyrinogen III, never form protoporphyrin IX, and GO currently has no molecular-function terms for the coproporphyrinogen oxidase (coproporphyrin-forming, EC 1.3.3.15), coproporphyrin ferrochelatase (EC 4.99.1.9) or coproheme decarboxylase (EC 1.3.98.5) reactions (OLS and QuickGO checked 2026-10-06). They belong in a sibling module once those terms exist. Family-level selectors here do not separate PPD enzymes from their CPD homologs: CgoX sits in the HemY/PPOX subfamily and CpfC in the ferrochelatase family. Membership of those families alone therefore does not show that a taxon uses the PPD route. A concrete organism or compartment may encode one or both ALA-entry systems and one or more enzymes on either late-oxidation axis; route enumeration describes minimal paths and therefore chooses one branch per axis. The catalog of late oxidation families is evidence-based but not a claim of universal cross-taxon completeness. ALIGN with YeastPathways (2026-10): the HEME-BIOSYNTHESIS-II, PWY3O-50 and heme part of PWY3O-69 frames map one-to-one onto the C4/Shemin entry, the shared trunk, the oxygen-dependent HemF route, the HemY/PPOX route and ferrochelatase. The eight YeastCyc catalysts (HEM1, HEM2, HEM3, HEM4, HEM12, HEM13, HEM14, HEM15) are correct and none are missing; S. cerevisiae and S. pombe orthologs were added as representatives. Fungi use only C4 entry, HemF and HemY. The MET1/MET8 siroheme branch of PWY3O-69 belongs to MODULE:siroheme_biosynthesis. Compartments in fungi: ALA synthase in the mitochondrial matrix, the trunk and coproporphyrinogen oxidase in the cytosol (unlike the intermembrane-space CPOX of animals), and protoporphyrinogen oxidase and ferrochelatase at the inner membrane. GO-CAM disagreements: YeastPathways_PWY3O-50 and _PWY3O-69 place HEM1 in the cytosol and PWY3O-69 also places HEM14 and HEM15 there, contradicting the mitochondrial localization; PWY3O-69 tags the siroheme activities as part of heme biosynthesis. The PomBase model agrees with the module chemistry and additionally contains transport, iron supply, heme A synthesis and a mitochondrial GSA aminomutase-like protein (SPCC417.11c, PANTHER PTHR43713) whose role is not established; fungi lack HemA, so it is not treated as C5-route evidence. Family and gene grounding (2026-11): every PANTHER family carried by a member protein now has a COMPLETE family review, including PTHR43013 (GluTR), PTHR43713 (GSAM), PTHR12390 and PTHR38042 (eukaryotic and prokaryotic/plastid uroporphyrinogen-III synthase), PTHR10755 (HemF/CPOX), PTHR13932 (HemN/HemW), PTHR40255 (HemJ) and PTHR38030 (HemG). The HemN family review scopes coproporphyrinogen dehydrogenase activity and porphyrin biosynthesis to the HemN subfamily (SF6), because the sister HemW/RSAD1 subfamily is a heme-chaperone lineage without that activity. The GSAM family review leaves GO:0042286 UNRESOLVED at family level, because PANTHER's main GSAM subfamily (SF3) also holds characterized intermolecular aminotransferases of aminoglycoside, lysine-fermentation, fumonisin and beta-phenylalanine metabolism; the module's GSAM step is therefore grounded on the proteobacterial PAINT node PTN000241400 (assessed SOUND), and the resulting FAMILY_FUNCTION_UNREVIEWED validator warning for the PTHR43713 family term is expected. The family term is kept because it records the verified membership of the representative GSAMs. The HemG family review (PTHR38030) likewise leaves GO:0070819 UNRESOLVED: its single subfamily also holds archaeal members from organisms that make heme without protoporphyrinogen IX, clostridial flavodoxins and the heme-degrading BilS, so the HemG step carries the same expected warning while its PANTHER subfamily term records the E. coli exemplar's verified membership. Every UniProt representative has a COMPLETE gene review. The ALAS and uroporphyrinogen-III synthase steps stay grounded to InterPro (IPR010961, IPR003754) because their members span several subfamilies of the broad PTHR13693 aminotransferase family and two separate PANTHER families (PTHR12390, PTHR38042) respectively; the module validator therefore cannot check the PAINT nodes under those steps for family consistency and says so. In the member gene reviews, a broad process row is MODIFY when the review proposes the specific replacement term in that row, and TOO_GENERAL when a more specific term the row's claim already entails is accepted on another row of the same review; both say the broad term is true.