adenosylcobalamin biosynthesis from adenosylcobinamide-GDP I

MetaCyc:PWY-5509

This three-reaction nucleotide-loop assembly pathway starts with supplied adenosylcobinamide-GDP and 5,6-dimethylbenzimidazole. CobT transfers phosphoribose from nicotinate ribonucleotide, CobS joins the resulting alpha-ribazole phosphate to the activated corrinoid, and CobC removes the terminal phosphate. The graph represents reviewed Escherichia coli K-12 assignments with explicit evidence limits: CobT homodimer and CobS function/magnesium annotations include similarity evidence, and the current E. coli CobC annotation is inferred from Salmonella CobC. K-12 growth genetics supports salvage; the 2021 EcCobS assays used a BL21-derived construct and heterologous accessory enzymes. A 2023 engineered cell-free B12 production system separately used MG1655 CobT, CobS and CobC, including a soluble SIMPLEx-CobS fusion and engineered complementation. This strengthens MG1655 functional support without establishing isolated CobC kinetics, native quaternary structures or terminal-step physiology. Chemical naming inconsistencies in that supplement are preserved as source conflicts; the graph uses independently balanced Rhea chemistry. Terminal-step physiological timing was reassessed in Salmonella, not directly in K-12. No universal enzyme essentiality, exclusive route or physiological flux is implied. The activated corrinoid input separates this exact pathway from upstream cobinamide uptake, reduction, adenosylation and GDP activation; this record does not cover the full salvage superpathway or de novo corrin-ring synthesis. The alternative unphosphorylated-ribazole route and cobamide remodeling are excluded.

Pathway type
cofactor biosynthesis
Organism scope

Read the source YAML

Pathway network

21 nodes · 25 asserted directed edges

Arrows show recorded subject → object relationships, not necessarily metabolic flow. Select a node for details or an arrow for evidence. Scroll, zoom or find a node to explore.

Pathway network: adenosylcobalamin biosynthesis from adenosylcobinamide-GDP I 21 nodes · 25 asserted directed edges. Every arrow links to its evidence table row; nodes link to their component details. Isolated declared components are retained. CHEBI:15890 — consumes → RHEA:11197; Evidence: RHEA:11197consumes CHEBI:57502 — consumes → RHEA:11197; Evidence: RHEA:11197consumes RHEA:11197 — produces → CHEBI:57918; Evidence: RHEA:11197produces RHEA:11197 — produces → CHEBI:32544; Evidence: RHEA:11197produces RHEA:11197 — produces → CHEBI:15378; Evidence: RHEA:11197produces CHEBI:57918 — consumes → RHEA:23561; Evidence: RHEA:23561consumes CHEBI:60487 — consumes → RHEA:23561; Evidence: RHEA:23561consumes RHEA:23561 — produces → CHEBI:60493; Evidence: RHEA:23561produces RHEA:23561 — produces → CHEBI:58115; Evidence: RHEA:23561produces RHEA:23561 — produces → CHEBI:15378; Evidence: RHEA:23561produces CHEBI:60493 — consumes → RHEA:30368; Evidence: RHEA:30368consumes CHEBI:15377 — consumes → RHEA:30368; Evidence: RHEA:30368consumes RHEA:30368 — produces → CHEBI:18408; Evidence: RHEA:30368produces RHEA:30368 — produces → CHEBI:43474; Evidence: RHEA:30368produces PathBank:SMP0001898/ProteinComplex/3870 — catalyzes → RHEA:11197; The native catalyst is represented as its CobT assembly, with the homodimer assignment qualified by similarity on the membership edge. The engineered 2023 system supports MG1655 enzyme function without establishing its native quaternary structure or physiological flux. The supplement misabbreviates nicotinate ribonucleotide as NMN; exact chemistry follows inspected Rhea and reviewed UniProt.; Evidence: PathBank:SMP0001898, UniProtKB:P36562, DOI:10.1038/s41467-023-40932-4catalyzes UniProtKB:P36562 — part_of → PathBank:SMP0001898/ProteinComplex/3870; Membership retains the native dimer coefficient without upgrading a similarity annotation to an experimentally established K-12 quaternary structure.; Evidence: PathBank:SMP0001898, UniProtKB:P36562part of UniProtKB:P36561 — enables → RHEA:23561; This gene-product/activity edge retains the reviewed K-12 assignment without asserting a physical monomer catalyst. The 2021 construct is BL21-derived; the 2023 work separately supports engineered MG1655 CobS function. Cyanation during product analysis limits inference about the native upper ligand from the final peak. The 2023 supplement misnames the product as adenosylcobinamide-5-P; the independently balanced Rhea equation is retained. No direct E. coli cobamide-remodeling activity is assigned.; Evidence: UniProtKB:P36561, MetaCyc:COBS-MONOMER, PMID:7592411, PMID:34031037, PMID:31609521, PMID:37620358, DOI:10.1038/s41467-023-40932-4enables UniProtKB:P52086 — enables → RHEA:30368; The reviewed K-12 annotation remains homology-qualified, with additional MG1655 functional support from the engineered 2023 multienzyme system. That system does not establish isolated CobC kinetics or native terminal-step physiology. Its supplement misnames the phosphate substrate and calls CobC[4] a synthase; exact chemistry follows independent Rhea and reviewed annotation. The P52086-linked 6E4B dimer is structurally assigned without solution-assembly evidence. No physical monomer state is inferred from the database frame name.; Evidence: UniProtKB:P52086, MetaCyc:RIBAZOLEPHOSPHAT-MONOMER, PMID:17209023, DOI:10.1038/s41467-023-40932-4, DOI:10.2210/pdb6E4B/pdbenables UniProtKB:P36561 — has_cofactor → CHEBI:18420; The magnesium requirement is retained as a similarity-qualified annotation. MgCl2 in the inspected reaction mixtures is an assay condition and is not an independent apo-enzyme metal-reconstitution or binding experiment.; Evidence: UniProtKB:P36561has cofactor UniProtKB:P36561 — located_in → GO:0005886; This retains the reviewed location assertion. The 2005 cited paper was inspected at abstract level; no detailed membrane topology or exclusive localization of every molecule is inferred.; Evidence: UniProtKB:P36561located in RHEA:11197 — part_of → MetaCyc:PWY-5509; Evidence: MetaCyc:PWY-5509, MetaCyc:DMBPPRIBOSYLTRANS-RXNpart of RHEA:23561 — part_of → MetaCyc:PWY-5509; Evidence: MetaCyc:PWY-5509, MetaCyc:COBALAMIN5PSYN-RXNpart of RHEA:30368 — part_of → MetaCyc:PWY-5509; Evidence: MetaCyc:PWY-5509, MetaCyc:RXN-8770, RHEA:30368part of RHEA:11197 — provides_input_for → RHEA:23561; The material-transfer edge represents the native annotated route. The cited 1999 reconstitution uses Salmonella enzymes; the later MG1655 proteins occur in a larger engineered multienzyme system, not an isolated native three-enzyme physiological-flux assay.; Evidence: MetaCyc:PWY-5509, PMID:10518530provides input for RHEA:23561 — provides_input_for → RHEA:30368; This sequence is the selected reviewed E. coli annotation with explicit cross-species inference in the CobC annotation and later engineered MG1655 system support. The earlier 1999 in vitro paper alone did not establish the physiological timing of phosphate removal; the 2007 reassessment establishes it specifically in Salmonella.; Evidence: MetaCyc:PWY-5509, PMID:10518530, PMID:17209023provides input for water; CHEBI:15377; Declared type: small_molecule small molecule water hydron; CHEBI:15378; Declared type: small_molecule small molecule hydron 5,6-dimethylbenzimidazole; CHEBI:15890; Declared type: small_molecule small molecule 5,6-dimethylbenzimidazole cobamamide; CHEBI:18408; Declared type: small_molecule small molecule cobamamide magnesium(2+); CHEBI:18420; Declared type: cofactor cofactor magnesium(2+) nicotinate; CHEBI:32544; Declared type: small_molecule small molecule nicotinate hydrogenphosphate; CHEBI:43474; Declared type: small_molecule small molecule hydrogenphosphate nicotinate D-ribonucleotide(2-); CHEBI:57502; Declared type: small_molecule small molecule nicotinate D-ribonucleotide(2-) alpha-ribazole 5'-phosphate(2-); CHEBI:57918; Declared type: small_molecule small molecule alpha-ribazole 5'-phosphate(2-) guanosine 5'-monophosphate(2-); CHEBI:58115; Declared type: small_molecule small molecule guanosine 5'-monophosphate(2-) adenosylcobinamide guanosyl diphosphate(1-); CHEBI:60487; Declared type: small_molecule small molecule adenosylcobinamide guanosyl diphosphate(1-) adenosylcobalamin 5'-phosphate(2-); CHEBI:60493; Declared type: small_molecule small molecule adenosylcobalamin 5'-phosphate(2-) Nicotinate-nucleotide--dimethylbenzimidazole phosphoribosyltransferase; UniProtKB:P36562; Declared type: protein protein Nicotinate-nucleotide-- dimethylbenzimidazole phosphoribosyltransferase Adenosylcobinamide-GDP ribazoletransferase; UniProtKB:P36561; Declared type: protein protein Adenosylcobinamide-GDP ribazoletransferase Adenosylcobalamin/alpha-ribazole phosphatase; UniProtKB:P52086; Declared type: protein protein Adenosylcobalamin/alpha- ribazole phosphatase nicotinate-nucleotide dimethylbenzimidazole phosphoribosyltransferase; PathBank:SMP0001898/ProteinComplex/3870; Declared type: complex complex nicotinate-nucleotide dimethylbenzimidazole phosphoribosyltransferase plasma membrane; GO:0005886; Declared type: cellular_component cellular component plasma membrane 5,6-dimethylbenzimidazole + nicotinate beta-D-ribonucleotide => alpha-ribazole 5'-phosphate + nicotinate + H(+); RHEA:11197; Declared type: molecular_activity; Source reaction direction: left_to_right molecular activity 5,6-dimethylbenzimidazole + nicotinate beta-D- ribonucleotide => alpha- ribazole 5'-phosphate + nicotinate + H(+) alpha-ribazole 5'-phosphate + adenosylcob(III)inamide-GDP => adenosylcob(III)alamin 5'-phosphate + GMP + H(+); RHEA:23561; Declared type: molecular_activity; Source reaction direction: left_to_right molecular activity alpha-ribazole 5'-phosphate + adenosylcob(III)inamide- GDP => adenosylcob(III)alamin 5'-phosphate + GMP + H(+) adenosylcob(III)alamin 5'-phosphate + H2O => adenosylcob(III)alamin + phosphate; RHEA:30368; Declared type: molecular_activity; Source reaction direction: left_to_right molecular activity adenosylcob(III)alamin 5'-phosphate + H2O => adenosylcob(III)alamin + phosphate adenosylcobalamin biosynthesis from adenosylcobinamide-GDP I; MetaCyc:PWY-5509; Declared type: pathway pathway adenosylcobalamin biosynthesis from adenosylcobinamide-GDP I

Components and activities

Declared biological components and source reaction directions
NodeKind or roleSource direction
Escherichia coli K-12
NCBITaxon:83333		taxon
water
CHEBI:15377		small molecule
hydron
CHEBI:15378		small molecule
5,6-dimethylbenzimidazole
CHEBI:15890		small molecule
cobamamide
CHEBI:18408		small molecule
magnesium(2+)
CHEBI:18420		cofactor
nicotinate
CHEBI:32544		small molecule
hydrogenphosphate
CHEBI:43474		small molecule
nicotinate D-ribonucleotide(2-)
CHEBI:57502		small molecule
alpha-ribazole 5'-phosphate(2-)
CHEBI:57918		small molecule
guanosine 5'-monophosphate(2-)
CHEBI:58115		small molecule
adenosylcobinamide guanosyl diphosphate(1-)
CHEBI:60487		small molecule
adenosylcobalamin 5'-phosphate(2-)
CHEBI:60493		small molecule
Nicotinate-nucleotide--dimethylbenzimidazole phosphoribosyltransferase
UniProtKB:P36562		protein
Adenosylcobinamide-GDP ribazoletransferase
UniProtKB:P36561		protein
Adenosylcobalamin/alpha-ribazole phosphatase
UniProtKB:P52086		protein
nicotinate-nucleotide dimethylbenzimidazole phosphoribosyltransferase
PathBank:SMP0001898/ProteinComplex/3870		complex
plasma membrane
GO:0005886		cellular component
5,6-dimethylbenzimidazole + nicotinate beta-D-ribonucleotide => alpha-ribazole 5'-phosphate + nicotinate + H(+)
RHEA:11197		molecular activityleft to right
alpha-ribazole 5'-phosphate + adenosylcob(III)inamide-GDP => adenosylcob(III)alamin 5'-phosphate + GMP + H(+)
RHEA:23561		molecular activityleft to right
adenosylcob(III)alamin 5'-phosphate + H2O => adenosylcob(III)alamin + phosphate
RHEA:30368		molecular activityleft to right

Mechanistic edges

Mechanistic edges and their cited evidence
SubjectPredicate and descriptionObjectEvidence
5,6-dimethylbenzimidazole
CHEBI:15890		consumes

5,6-dimethylbenzimidazole + nicotinate beta-D-ribonucleotide => alpha-ribazole 5'-phosphate + nicotinate + H(+)
RHEA:11197
  • RHEA:11197

    Source assertion: Rhea RHEA:11197 assigns CHEBI:15890 to its substrates with coefficient 1; the exact charged species is linked through rh:compound/rh:chebi.

    Source locationrdf:Description[@rdf:about='http://rdf.rhea-db.org/11197']/rh:substrates; rdf:Description[@rdf:about='http://rdf.rhea-db.org/11196_L']/rh:contains1[@rdf:resource='http://rdf.rhea-db.org/Participant_11196_compound_2241']; rh:compound/rh:chebi
nicotinate D-ribonucleotide(2-)
CHEBI:57502		consumes

5,6-dimethylbenzimidazole + nicotinate beta-D-ribonucleotide => alpha-ribazole 5'-phosphate + nicotinate + H(+)
RHEA:11197
  • RHEA:11197

    Source assertion: Rhea RHEA:11197 assigns CHEBI:57502 to its substrates with coefficient 1; the exact charged species is linked through rh:compound/rh:chebi.

    Source locationrdf:Description[@rdf:about='http://rdf.rhea-db.org/11197']/rh:substrates; rdf:Description[@rdf:about='http://rdf.rhea-db.org/11196_L']/rh:contains1[@rdf:resource='http://rdf.rhea-db.org/Participant_11196_compound_2274']; rh:compound/rh:chebi
5,6-dimethylbenzimidazole + nicotinate beta-D-ribonucleotide => alpha-ribazole 5'-phosphate + nicotinate + H(+)
RHEA:11197		produces

alpha-ribazole 5'-phosphate(2-)
CHEBI:57918
  • RHEA:11197

    Source assertion: Rhea RHEA:11197 assigns CHEBI:57918 to its products with coefficient 1; the exact charged species is linked through rh:compound/rh:chebi.

    Source locationrdf:Description[@rdf:about='http://rdf.rhea-db.org/11197']/rh:products; rdf:Description[@rdf:about='http://rdf.rhea-db.org/11196_R']/rh:contains1[@rdf:resource='http://rdf.rhea-db.org/Participant_11196_compound_1950']; rh:compound/rh:chebi
5,6-dimethylbenzimidazole + nicotinate beta-D-ribonucleotide => alpha-ribazole 5'-phosphate + nicotinate + H(+)
RHEA:11197		produces

nicotinate
CHEBI:32544
  • RHEA:11197

    Source assertion: Rhea RHEA:11197 assigns CHEBI:32544 to its products with coefficient 1; the exact charged species is linked through rh:compound/rh:chebi.

    Source locationrdf:Description[@rdf:about='http://rdf.rhea-db.org/11197']/rh:products; rdf:Description[@rdf:about='http://rdf.rhea-db.org/11196_R']/rh:contains1[@rdf:resource='http://rdf.rhea-db.org/Participant_11196_compound_2096']; rh:compound/rh:chebi
5,6-dimethylbenzimidazole + nicotinate beta-D-ribonucleotide => alpha-ribazole 5'-phosphate + nicotinate + H(+)
RHEA:11197		produces

hydron
CHEBI:15378
  • RHEA:11197

    Source assertion: Rhea RHEA:11197 assigns CHEBI:15378 to its products with coefficient 1; the exact charged species is linked through rh:compound/rh:chebi.

    Source locationrdf:Description[@rdf:about='http://rdf.rhea-db.org/11197']/rh:products; rdf:Description[@rdf:about='http://rdf.rhea-db.org/11196_R']/rh:contains1[@rdf:resource='http://rdf.rhea-db.org/Participant_11196_compound_3249']; rh:compound/rh:chebi
alpha-ribazole 5'-phosphate(2-)
CHEBI:57918		consumes

alpha-ribazole 5'-phosphate + adenosylcob(III)inamide-GDP => adenosylcob(III)alamin 5'-phosphate + GMP + H(+)
RHEA:23561
  • RHEA:23561

    Source assertion: Rhea RHEA:23561 assigns CHEBI:57918 to its substrates with coefficient 1; the exact charged species is linked through rh:compound/rh:chebi.

    Source locationrdf:Description[@rdf:about='http://rdf.rhea-db.org/23561']/rh:substrates; rdf:Description[@rdf:about='http://rdf.rhea-db.org/23560_L']/rh:contains1[@rdf:resource='http://rdf.rhea-db.org/Participant_23560_compound_1950']; rh:compound/rh:chebi
adenosylcobinamide guanosyl diphosphate(1-)
CHEBI:60487		consumes

alpha-ribazole 5'-phosphate + adenosylcob(III)inamide-GDP => adenosylcob(III)alamin 5'-phosphate + GMP + H(+)
RHEA:23561
  • RHEA:23561

    Source assertion: Rhea RHEA:23561 assigns CHEBI:60487 to its substrates with coefficient 1; the exact charged species is linked through rh:compound/rh:chebi.

    Source locationrdf:Description[@rdf:about='http://rdf.rhea-db.org/23561']/rh:substrates; rdf:Description[@rdf:about='http://rdf.rhea-db.org/23560_L']/rh:contains1[@rdf:resource='http://rdf.rhea-db.org/Participant_23560_compound_6530']; rh:compound/rh:chebi
alpha-ribazole 5'-phosphate + adenosylcob(III)inamide-GDP => adenosylcob(III)alamin 5'-phosphate + GMP + H(+)
RHEA:23561		produces

adenosylcobalamin 5'-phosphate(2-)
CHEBI:60493
  • RHEA:23561

    Source assertion: Rhea RHEA:23561 assigns CHEBI:60493 to its products with coefficient 1; the exact charged species is linked through rh:compound/rh:chebi.

    Source locationrdf:Description[@rdf:about='http://rdf.rhea-db.org/23561']/rh:products; rdf:Description[@rdf:about='http://rdf.rhea-db.org/23560_R']/rh:contains1[@rdf:resource='http://rdf.rhea-db.org/Participant_23560_compound_6640']; rh:compound/rh:chebi
alpha-ribazole 5'-phosphate + adenosylcob(III)inamide-GDP => adenosylcob(III)alamin 5'-phosphate + GMP + H(+)
RHEA:23561		produces

guanosine 5'-monophosphate(2-)
CHEBI:58115
  • RHEA:23561

    Source assertion: Rhea RHEA:23561 assigns CHEBI:58115 to its products with coefficient 1; the exact charged species is linked through rh:compound/rh:chebi.

    Source locationrdf:Description[@rdf:about='http://rdf.rhea-db.org/23561']/rh:products; rdf:Description[@rdf:about='http://rdf.rhea-db.org/23560_R']/rh:contains1[@rdf:resource='http://rdf.rhea-db.org/Participant_23560_compound_3807']; rh:compound/rh:chebi
alpha-ribazole 5'-phosphate + adenosylcob(III)inamide-GDP => adenosylcob(III)alamin 5'-phosphate + GMP + H(+)
RHEA:23561		produces

hydron
CHEBI:15378
  • RHEA:23561

    Source assertion: Rhea RHEA:23561 assigns CHEBI:15378 to its products with coefficient 1; the exact charged species is linked through rh:compound/rh:chebi.

    Source locationrdf:Description[@rdf:about='http://rdf.rhea-db.org/23561']/rh:products; rdf:Description[@rdf:about='http://rdf.rhea-db.org/23560_R']/rh:contains1[@rdf:resource='http://rdf.rhea-db.org/Participant_23560_compound_3249']; rh:compound/rh:chebi
adenosylcobalamin 5'-phosphate(2-)
CHEBI:60493		consumes

adenosylcob(III)alamin 5'-phosphate + H2O => adenosylcob(III)alamin + phosphate
RHEA:30368
  • RHEA:30368

    Source assertion: Rhea RHEA:30368 assigns CHEBI:60493 to its substrates with coefficient 1; the exact charged species is linked through rh:compound/rh:chebi.

    Source locationrdf:Description[@rdf:about='http://rdf.rhea-db.org/30368']/rh:substrates; rdf:Description[@rdf:about='http://rdf.rhea-db.org/30367_L']/rh:contains1[@rdf:resource='http://rdf.rhea-db.org/Participant_30367_compound_6640']; rh:compound/rh:chebi
water
CHEBI:15377		consumes

adenosylcob(III)alamin 5'-phosphate + H2O => adenosylcob(III)alamin + phosphate
RHEA:30368
  • RHEA:30368

    Source assertion: Rhea RHEA:30368 assigns CHEBI:15377 to its substrates with coefficient 1; the exact charged species is linked through rh:compound/rh:chebi.

    Source locationrdf:Description[@rdf:about='http://rdf.rhea-db.org/30368']/rh:substrates; rdf:Description[@rdf:about='http://rdf.rhea-db.org/30367_L']/rh:contains1[@rdf:resource='http://rdf.rhea-db.org/Participant_30367_compound_1283']; rh:compound/rh:chebi
adenosylcob(III)alamin 5'-phosphate + H2O => adenosylcob(III)alamin + phosphate
RHEA:30368		produces

cobamamide
CHEBI:18408
  • RHEA:30368

    Source assertion: Rhea RHEA:30368 assigns CHEBI:18408 to its products with coefficient 1; the exact charged species is linked through rh:compound/rh:chebi.

    Source locationrdf:Description[@rdf:about='http://rdf.rhea-db.org/30368']/rh:products; rdf:Description[@rdf:about='http://rdf.rhea-db.org/30367_R']/rh:contains1[@rdf:resource='http://rdf.rhea-db.org/Participant_30367_compound_4165']; rh:compound/rh:chebi
adenosylcob(III)alamin 5'-phosphate + H2O => adenosylcob(III)alamin + phosphate
RHEA:30368		produces

hydrogenphosphate
CHEBI:43474
  • RHEA:30368

    Source assertion: Rhea RHEA:30368 assigns CHEBI:43474 to its products with coefficient 1; the exact charged species is linked through rh:compound/rh:chebi.

    Source locationrdf:Description[@rdf:about='http://rdf.rhea-db.org/30368']/rh:products; rdf:Description[@rdf:about='http://rdf.rhea-db.org/30367_R']/rh:contains1[@rdf:resource='http://rdf.rhea-db.org/Participant_30367_compound_5518']; rh:compound/rh:chebi
nicotinate-nucleotide dimethylbenzimidazole phosphoribosyltransferase
PathBank:SMP0001898/ProteinComplex/3870		catalyzes

The native catalyst is represented as its CobT assembly, with the homodimer assignment qualified by similarity on the membership edge. The engineered 2023 system supports MG1655 enzyme function without establishing its native quaternary structure or physiological flux. The supplement misabbreviates nicotinate ribonucleotide as NMN; exact chemistry follows inspected Rhea and reviewed UniProt.

5,6-dimethylbenzimidazole + nicotinate beta-D-ribonucleotide => alpha-ribazole 5'-phosphate + nicotinate + H(+)
RHEA:11197
  • PathBank:SMP0001898

    Source assertion: Native ReactionCatalysis/5115 uses ProteinComplex/3870 as controller of Reactions/5142, the DMB plus nicotinate-ribonucleotide reaction producing alpha-ribazole phosphate, nicotinate and a proton.

    Source locationPW001884.owl: bp:Catalysis[@rdf:ID="ReactionCatalysis/5115"]/bp:controller; bp:controlled; bp:BiochemicalReaction[@rdf:ID="Reactions/5142"]/bp:left; bp:right; bp:name
  • UniProtKB:P36562

    Source assertion: Reviewed K-12 CobT P36562 assigns DMB plus nicotinate beta-D-ribonucleotide to alpha-ribazole 5'-phosphate, nicotinate and H(+), with RHEA:11196. The reaction comment has no inline experimental qualifier.

    Source location/primaryAccession; /organism/taxonId; /comments/0; /comments/1/reaction
  • DOI:10.1038/s41467-023-40932-4

    Source assertion: The 2023 supplement identifies the CobT used in the engineered cell-free B12 synthesis system as E. coli MG1655 EC 2.4.2.21. This supports use of the MG1655 enzyme in a multienzyme system, not direct characterization of its dimer.

    Source locationSupplementary Information, Supplementary Table 1, PDF page 4; Supplementary Table 2, PDF pages 7-9; main paper Methods, Cloning and purification of enzymes
Nicotinate-nucleotide--dimethylbenzimidazole phosphoribosyltransferase
UniProtKB:P36562		part_of

Membership retains the native dimer coefficient without upgrading a similarity annotation to an experimentally established K-12 quaternary structure.

nicotinate-nucleotide dimethylbenzimidazole phosphoribosyltransferase
PathBank:SMP0001898/ProteinComplex/3870
  • PathBank:SMP0001898

    Source assertion: ProteinComplex/3870 contains Protein/6552 with coefficient 2.0. Protein/6552 resolves through ProteinReference/6552 and Reference/UniProt_P36562 to accession P36562.

    Source locationPW001884.owl: bp:Complex[@rdf:ID="ProteinComplex/3870"]/bp:component; bp:componentStoichiometry; bp:Stoichiometry[@rdf:ID="Protein/6552/Stoichiometry/2.0"]; bp:ProteinReference[@rdf:ID="ProteinReference/6552"]/bp:xref; bp:UnificationXref[@rdf:ID="Reference/UniProt_P36562"]
  • UniProtKB:P36562

    Source assertion: Reviewed CobT is annotated as a homodimer with ECO:0000250, an inference by similarity.

    Source location/comments/3/commentType; /comments/3/texts/0/value; /comments/3/texts/0/evidences/0/evidenceCode
Adenosylcobinamide-GDP ribazoletransferase
UniProtKB:P36561		enables

This gene-product/activity edge retains the reviewed K-12 assignment without asserting a physical monomer catalyst. The 2021 construct is BL21-derived; the 2023 work separately supports engineered MG1655 CobS function. Cyanation during product analysis limits inference about the native upper ligand from the final peak. The 2023 supplement misnames the product as adenosylcobinamide-5-P; the independently balanced Rhea equation is retained. No direct E. coli cobamide-remodeling activity is assigned.

alpha-ribazole 5'-phosphate + adenosylcob(III)inamide-GDP => adenosylcob(III)alamin 5'-phosphate + GMP + H(+)
RHEA:23561
  • UniProtKB:P36561

    Source assertion: Reviewed K-12 CobS includes the alpha-ribazole 5'-phosphate plus adenosylcobinamide-GDP reaction RHEA:23560. Its FUNCTION comment explicitly qualifies this synthesis by similarity (ECO:0000250).

    Source location/primaryAccession; /organism/taxonId; /comments/0; /comments/2/reaction
  • MetaCyc:COBS-MONOMER

    Source assertion: COBS-MONOMER maps to P36561 and catalyzes COBALAMIN5PSYN-RXN through COBALAMIN5PSYN-ENZRXN. The native comment cites Salmonella terminal-step reassessment for the function.

    Source locationProtein[@frameid="COBS-MONOMER"]/dblink[dblink-db="UNIPROT"]; catalyzes/Enzymatic-Reaction[@frameid="COBALAMIN5PSYN-ENZRXN"]; comment
  • PMID:7592411

    Source assertion: E. coli K-12 cobS mutants failed the cobinamide-dependent growth test even with DMB supplementation. This supports salvage function, not a purified assay of each current Rhea species.

    Source locationPMC177486: Results, Localization of mutations within the E. coli cob operon; Tables 2 and 3, printed page 6375; Materials and Methods, strain and cloning scope
  • PMID:34031037

    Source assertion: EcCobS-containing membranes supported the cobinamide-salvaging control with alpha-ribazole phosphate, Salmonella CobU and Vibrio CobC. The experiment used a BL21-derived host and does not assay K-12 CobC.

    Source locationPMC8407341: Results, In vitro reactions for VcCobS remodeling; Figure 4A; Table 1; Materials and Methods, In vitro cobamide production assays
  • PMID:31609521

    Source assertion: The EcCobS expression construct was cloned from BL21. This identifies the origin of the construct subsequently cited in the 2021 paper; exact equivalence to K-12 accession P36561 is not established here.

    Source locationExperimental procedures, Strain cultivation and cloning, second paragraph; pET16b EcCobS construct
  • PMID:37620358

    Source assertion: The 2023 study cloned CobS from E. coli MG1655, fused it to ApoAI* and signal-peptide-deleted MBP for the SIMPLEx system, and tested engineered B12 production and a cell-free cascade. This is an MG1655 construct with engineered solubilization, not a native membrane assembly assay.

    Source locationPMC10449867: Results, Promoting the reactions catalyzed by transmembrane enzymes; Methods, Cloning and purification of enzymes; Supplementary Figure 9d
  • DOI:10.1038/s41467-023-40932-4

    Source assertion: Supplementary Table 1 identifies CobS as E. coli MG1655 EC 2.7.8.26; Supplementary Figure 9d compares the engineered B16 parent, cobS deletion, and CobS or SIMPLEx-CobS complementation. Its reaction product name is inconsistent with the intended cobalamin-phosphate structure.

    Source locationSupplementary Information, Supplementary Table 1, PDF page 4; Supplementary Figure 9d and legend, PDF page 23
Adenosylcobalamin/alpha-ribazole phosphatase
UniProtKB:P52086		enables

The reviewed K-12 annotation remains homology-qualified, with additional MG1655 functional support from the engineered 2023 multienzyme system. That system does not establish isolated CobC kinetics or native terminal-step physiology. Its supplement misnames the phosphate substrate and calls CobC[4] a synthase; exact chemistry follows independent Rhea and reviewed annotation. The P52086-linked 6E4B dimer is structurally assigned without solution-assembly evidence. No physical monomer state is inferred from the database frame name.

adenosylcob(III)alamin 5'-phosphate + H2O => adenosylcob(III)alamin + phosphate
RHEA:30368
  • UniProtKB:P52086

    Source assertion: Reviewed K-12 CobC assigns adenosylcobalamin 5'-phosphate hydrolysis to RHEA:30367, with ECO:0000250 and UniProtKB:P39701 as the homology source.

    Source location/primaryAccession; /organism/taxonId; /comments/0; /comments/1/reaction/evidences
  • MetaCyc:RIBAZOLEPHOSPHAT-MONOMER

    Source assertion: The version-22.5 P52086 frame describes E. coli K-12 CobC as uncharacterized and predicts its phosphatase function by Salmonella homology. This historical source statement does not account for the subsequently inspected 2023 engineered MG1655 system.

    Source locationProtein[@frameid="RIBAZOLEPHOSPHAT-MONOMER"]/comment; dblink[dblink-db="UNIPROT"]; catalyzes/Enzymatic-Reaction[@frameid="ENZRXN0-7469"]
  • PMID:17209023

    Source assertion: Salmonella Typhimurium cobC mutants accumulated cobalamin phosphate; the paper assigns terminal dephosphorylation to CobC and explicitly limits this physiological conclusion to that organism.

    Source locationPMC1899380: Results, A cobC strain accumulates AdoCbl-P; Figures 5 and 6; Discussion, opening paragraph; Figure 8
  • DOI:10.1038/s41467-023-40932-4

    Source assertion: Supplementary Table 1 identifies the CobC phosphatase used in the 2023 cell-free system as E. coli MG1655 EC 3.1.3.73. Table 2 includes this enzyme as CobC[4]. This is multienzyme-system support, not isolated CobC kinetics; the reported K′eq column is calculated thermodynamics.

    Source locationSupplementary Information, Supplementary Table 1, PDF pages 2 and 5-6; Supplementary Table 2 and footnote [4], PDF pages 7-9; main paper Methods, Cloning and purification of enzymes
  • DOI:10.2210/pdb6E4B/pdb

    Source assertion: RCSB assembly 1 for 6E4B is an author-and-PISA-defined dimer. The author-evidence field explicitly reports experimental_support as none; this does not establish a separately measured solution oligomerization state.

    Source locationRCSB core assembly/6E4B/1 JSON: /pdbx_struct_assembly; /pdbx_struct_assembly_auth_evidence/0/experimental_support; /rcsb_assembly_info
Adenosylcobinamide-GDP ribazoletransferase
UniProtKB:P36561		has_cofactor

The magnesium requirement is retained as a similarity-qualified annotation. MgCl2 in the inspected reaction mixtures is an assay condition and is not an independent apo-enzyme metal-reconstitution or binding experiment.

magnesium(2+)
CHEBI:18420
  • UniProtKB:P36561

    Source assertion: Reviewed CobS names Mg(2+) as cofactor, linked to CHEBI:18420, with ECO:0000250.

    Source location/comments/3/commentType; /comments/3/cofactors/0
Adenosylcobinamide-GDP ribazoletransferase
UniProtKB:P36561		located_in

This retains the reviewed location assertion. The 2005 cited paper was inspected at abstract level; no detailed membrane topology or exclusive localization of every molecule is inferred.

plasma membrane
GO:0005886
  • UniProtKB:P36561

    Source assertion: Reviewed P36561 has cell-inner-membrane, multipass annotation with ECO:0000269 citing PMID:15919996, and an explicit GO:0005886 cross-reference with IDA:EcoCyc and ECO:0000314.

    Source location/comments/5/subcellularLocations/0; /uniProtKBCrossReferences/32
5,6-dimethylbenzimidazole + nicotinate beta-D-ribonucleotide => alpha-ribazole 5'-phosphate + nicotinate + H(+)
RHEA:11197		part_of

MetaCyc:PWY-5509
  • MetaCyc:PWY-5509

    Source assertion: PWY-5509 lists DMBPPRIBOSYLTRANS-RXN as a member and selects L2R in reaction-layout.

    Source locationPathway[@frameid="PWY-5509"]/reaction-list/Reaction[@frameid="DMBPPRIBOSYLTRANS-RXN"]; reaction-layout[Reaction[@frameid="DMBPPRIBOSYLTRANS-RXN"]]
  • MetaCyc:DMBPPRIBOSYLTRANS-RXN

    Source assertion: DMBPPRIBOSYLTRANS-RXN supplies the matching chemical sides and the directional RHEA:11197 cross-reference.

    Source locationReaction/left; right; reaction-direction; dblink[dblink-db="RHEA"]
alpha-ribazole 5'-phosphate + adenosylcob(III)inamide-GDP => adenosylcob(III)alamin 5'-phosphate + GMP + H(+)
RHEA:23561		part_of

MetaCyc:PWY-5509
  • MetaCyc:PWY-5509

    Source assertion: PWY-5509 lists COBALAMIN5PSYN-RXN as a member and selects L2R in reaction-layout.

    Source locationPathway[@frameid="PWY-5509"]/reaction-list/Reaction[@frameid="COBALAMIN5PSYN-RXN"]; reaction-layout[Reaction[@frameid="COBALAMIN5PSYN-RXN"]]
  • MetaCyc:COBALAMIN5PSYN-RXN

    Source assertion: COBALAMIN5PSYN-RXN supplies the matching chemical sides and the directional RHEA:23561 cross-reference.

    Source locationReaction/left; right; reaction-direction; dblink[dblink-db="RHEA"]
adenosylcob(III)alamin 5'-phosphate + H2O => adenosylcob(III)alamin + phosphate
RHEA:30368		part_of

MetaCyc:PWY-5509
  • MetaCyc:PWY-5509

    Source assertion: PWY-5509 lists RXN-8770 as a member and selects L2R in reaction-layout.

    Source locationPathway[@frameid="PWY-5509"]/reaction-list/Reaction[@frameid="RXN-8770"]; reaction-layout[Reaction[@frameid="RXN-8770"]]
  • MetaCyc:RXN-8770

    Source assertion: RXN-8770 supplies the hydrolysis sides, a bidirectional RHEA:30370 cross-reference and a PHYSIOL-LEFT-TO-RIGHT direction. Selection of RHEA:30368 additionally uses the separately cited Rhea family.

    Source locationReaction/left; right; reaction-direction; dblink[dblink-db="RHEA"]
  • RHEA:30368

    Source assertion: Rhea master 30367 links bidirectional reaction 30370 and directional reaction 30368. Reaction 30368 consumes master side 30367_L and produces 30367_R, selecting the hydrolysis direction that matches native RXN-8770.

    Source locationrdf:Description[@rdf:about="http://rdf.rhea-db.org/30367"]/rh:bidirectionalReaction; rh:directionalReaction; rdf:Description[@rdf:about="http://rdf.rhea-db.org/30368"]/rh:substrates; rh:products
5,6-dimethylbenzimidazole + nicotinate beta-D-ribonucleotide => alpha-ribazole 5'-phosphate + nicotinate + H(+)
RHEA:11197		provides_input_for

The material-transfer edge represents the native annotated route. The cited 1999 reconstitution uses Salmonella enzymes; the later MG1655 proteins occur in a larger engineered multienzyme system, not an isolated native three-enzyme physiological-flux assay.

alpha-ribazole 5'-phosphate + adenosylcob(III)inamide-GDP => adenosylcob(III)alamin 5'-phosphate + GMP + H(+)
RHEA:23561
  • MetaCyc:PWY-5509

    Source assertion: PWY-5509 orders DMBPPRIBOSYLTRANS-RXN before COBALAMIN5PSYN-RXN. Alpha-ribazole 5'-phosphate is the first reaction's product and the second reaction's substrate.

    Source locationPathway[@frameid="PWY-5509"]/reaction-ordering[Reaction[@frameid="COBALAMIN5PSYN-RXN"]]/predecessor-reactions; reaction-layout
  • PMID:10518530

    Source assertion: The Salmonella enzyme reconstitution uses the CobT product alpha-ribazole 5'-phosphate with adenosylcobinamide-GDP for CobS-dependent formation of cobalamin phosphate.

    Source locationPMC18366: Results, The CobS Protein Has Cobalamin Synthase Activity; Isolation of Products from the NLA and Cobalamin Synthase Reactions; Figures 3C and 4
alpha-ribazole 5'-phosphate + adenosylcob(III)inamide-GDP => adenosylcob(III)alamin 5'-phosphate + GMP + H(+)
RHEA:23561		provides_input_for

This sequence is the selected reviewed E. coli annotation with explicit cross-species inference in the CobC annotation and later engineered MG1655 system support. The earlier 1999 in vitro paper alone did not establish the physiological timing of phosphate removal; the 2007 reassessment establishes it specifically in Salmonella.

adenosylcob(III)alamin 5'-phosphate + H2O => adenosylcob(III)alamin + phosphate
RHEA:30368
  • MetaCyc:PWY-5509

    Source assertion: PWY-5509 orders RXN-8770 after COBALAMIN5PSYN-RXN through the adenosylcobalamin 5'-phosphate intermediate.

    Source locationPathway[@frameid="PWY-5509"]/reaction-ordering[Reaction[@frameid="RXN-8770"]]/predecessor-reactions; reaction-layout
  • PMID:10518530

    Source assertion: Salmonella CobC-containing extract dephosphorylated the cobalamin phosphate formed by nucleotide-loop assembly; vector-only extract did not. Corrinoids were cyanated before HPLC analysis.

    Source locationPMC18366: Results, The CobC Protein Can Dephosphorylate AdoCbl-5'-P in Vitro; Figure 6
  • PMID:17209023

    Source assertion: The later Salmonella study supports alpha-ribazole phosphate use by CobS followed by terminal CobC dephosphorylation. Its Discussion states that validity in other prokaryotes remains to be shown.

    Source locationPMC1899380: Results, Alpha-RP is the preferred substrate of the AdoCbl-5'-P synthase (CobS) enzyme; Figure 7; Discussion, opening paragraph; Figure 8

References

Curation history