De novo riboflavin biosynthesis and flavin cofactor activation

De novo synthesis of riboflavin (vitamin B2) from GTP and ribulose 5-phosphate, followed by conversion of riboflavin to the active flavin cofactors FMN and FAD, as found in bacteria, fungi and plants. The ring pathway has two converging branches. In the GTP-derived pyrimidine branch, GTP cyclohydrolase II opens the imidazole ring of GTP; the product then undergoes deamination of the pyrimidine ring and reduction of the ribosyl side chain, and the resulting phosphorylated intermediate is dephosphorylated. The order of deamination and reduction differs by lineage: bacteria deaminate first and then reduce, usually with one bifunctional RibD protein, whereas fungi reduce first and then deaminate, with two separate proteins. The ribulose-5-phosphate branch makes the four-carbon donor 3,4-dihydroxy-2-butanone 4-phosphate. The two branches converge at lumazine synthase, and riboflavin synthase then forms riboflavin by a dismutation of two lumazine molecules. Riboflavin kinase converts riboflavin to FMN and FMN adenylyltransferase converts FMN to FAD. In many bacteria both activation steps are carried out by one bifunctional RibF protein, whereas eukaryotes use a monofunctional riboflavin kinase and a separate FAD synthase.

The module deliberately separates riboflavin-ring synthesis from general flavoprotein use, FMN-dependent reductases, prenylated-FMN cofactor formation, and cobalamin lower-ligand biosynthesis. Those processes consume or transform flavins but are not required steps in de novo riboflavin formation. The downstream FMN/FAD activation step is included because bacterial RibF-family enzymes often connect riboflavin synthesis directly to active flavin-cofactor supply; animal-only uptake of dietary riboflavin is covered separately by modules/flavin_cofactor_biosynthesis.yaml and modules/riboflavin_uptake_and_cofactor_synthesis.yaml. Human RFK and FLAD1 are included below only as cross-organism representatives of the shared activation activities, not as members of a de novo route. In the Pseudomonas putida KT2440 instantiation, ribAB-I (PP_0516) and ribAB-II (PP_3813) are RibBX-type DHBP synthases: their C-terminal GTP cyclohydrolase II-like domains lack the zinc-binding cysteines and the catalytic arginine of RibA (gene-review active-site check; PMID:24097946), so this module uses them only as DHBP synthase exemplars and the KT2440 gene reviews remove their TreeGrafter GTP cyclohydrolase II rows. KT2440 RibA (PP_0522) is the single GTP cyclohydrolase II, and ribE (PP_0515) and ribC (PP_2916) are two intact riboflavin synthase paralogs. Note the symbol clash with E. coli, where ribE is lumazine synthase and ribC riboflavin synthase. Family grounding: in PTHR21327 (subfamily SF18) and PTHR22749 (subfamily SF6) monofunctional and bifunctional members share a subfamily, so the family reviews leave DHBP synthase and bacterial FMN adenylyltransferase activity UNRESOLVED at the family level, and the bacterial RibD deaminase is UNRESOLVED in PTHR38011 (whole bifunctional RibD proteins share SF7 with stand-alone fungal reductases). These annotons are therefore grounded on gene-reviewed representatives, and the verified PANTHER ids are kept as membership records. GTP cyclohydrolase II in PTHR21327 is scoped RESIDUE_DETERMINED by the RibA zinc-triad/catalytic site (gtpchii_catalytic_core). Gene-review completeness is 27/28: human FLAD1 is left at computed status DRAFT because, under the ClinGen Mendelian project's standing instruction, its supported bare protein-binding rows are kept as non-core and the generic protein-binding policy warns on them; FLAD1 is only a cross-organism representative of the eukaryotic FAD synthase step. Fungal alignment (YeastPathways YEAST-RIBOSYN-PWY and the PomBase model 6796b94c00000731): the deamination/reduction pair is modelled as an order variant set. GO has no separate terms for the fungal-order substrates: GO:0043723 (deamination of the ribityl intermediate) was made obsolete and merged into GO:0008835, and GO:0008703 is defined on the bacterial-order (deaminated) substrate although the fungal reductase acts on the non-deaminated intermediate (EC 1.1.1.302 rather than EC 1.1.1.193). The fungal annotons therefore use GO:0008835 and GO:0008703, as SGD and PomBase do, and record the substrate difference in the function description. The fungal deaminase domain is fused to unrelated N-terminal domains (a tRNA pseudouridine synthase in S. cerevisiae RIB2, a lysine methyltransferase in S. pombe rib2), so no single PANTHER family is asserted for it. GO-CAM notes: the SGD model types RIB2 (RXN3O-164) only with the root term GO:0003674 and places all activities in cytosol; the PomBase model also includes urg1 as a second GTP cyclohydrolase II. urg1 is a uracil-regulated protein in a different PANTHER family (PTHR47259) and is not used here as a riboflavin pathway exemplar; the PomBase model's mitochondrial FAD transporter flx1 lies outside this module boundary. Yeast reduction-before-deamination is also reported in PMID:42017481. The RibT N-acetyltransferase, proposed to regulate lumazine synthase in Bacillus subtilis, is regulatory context and not a pathway step.