id: P26165 gene_symbol: bchF product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:272942 label: Rhodobacter capsulatus SB 1003 description: >- bchF encodes the 3-vinyl (bacterio)chlorophyllide a hydratase of the purple non-sulfur bacterium Rhodobacter capsulatus, a small (171-residue), strongly hydrophobic enzyme encoded in the 46 kb photosynthesis gene cluster next to bchN, bchB and bchH. It catalyses one step of the bacteriochlorophyll a-specific branch of chlorophyll metabolism: addition of water across the C-3 vinyl group of chlorophyllide a or of 3-vinyl bacteriochlorophyllide a to give the 3-(1-hydroxyethyl) intermediate, which the dehydrogenase BchC then oxidises to the 3-acetyl group that distinguishes bacteriochlorophyll a from chlorophyll a. The pathway it serves operates independently of light, since protochlorophyllide reduction in these bacteria is carried out by the dark-operative BchLNB enzyme. Transposon-insertion and directed deletion mutants of bchF lose bacteriochlorophyll a and accumulate the unhydrated precursors chlorophyllide a and 3-vinyl bacteriochlorophyllide a, the latter of which can still be phytylated and assembled into pigment-protein complexes. Purified orthologues from green sulfur and other chlorophototrophic bacteria hydrate the C-3 vinyl group in vitro and run the reaction reversibly, so the family is a genuine, stereoselective hydratase/dehydratase rather than a structural component of the pathway. The protein itself has not been purified from R. capsulatus and its membrane topology is undetermined. references: - id: GO_REF:0000002 title: Gene Ontology annotation through association of InterPro records with GO terms findings: - statement: >- The InterPro2GO mapping file assigns three GO terms to IPR009905 (2-vinyl bacteriochlorophyllide hydratase): GO:0016836 hydro-lyase activity, GO:0030494 bacteriochlorophyll biosynthetic process, and GO:0019685 photosynthesis, dark reaction. The first two fit the family; the third does not, because GO:0019685 is the carbon-fixation grouping term - its children are the Calvin cycle, C4 and CAM photosynthesis - and not the light-independent segment of the bacteriochlorophyll pathway. All four InterPro2GO rules that use GO:0019685 are light-independent (bacterio)chlorophyll-synthesis families, and QuickGO reports that all 3,731 GOA annotations to GO:0019685 cite GO_REF:0000002, so every use of the term in GOA descends from this one misreading (checked 2026-11-01). reference_review: relevance: HIGH correctness: MISCITED review_notes: >- The GO_REF itself is a valid method reference; the defect is in one of the three IPR009905 mappings it delivers (GO:0019685), recorded as a source_defect on that annotation row. - id: GO_REF:0000041 title: Gene Ontology annotation based on UniPathway vocabulary mapping findings: - statement: >- UniProt maps the bchF entry to UniPathway UPA00671, porphyrin-containing compound metabolism / bacteriochlorophyll biosynthesis (light-independent), which yields GO:0036070. The pathway assignment agrees with the genetics: in R. capsulatus protochlorophyllide reduction is carried out in the dark by BchL/BchN/BchB. reference_review: relevance: HIGH correctness: VERIFIED review_notes: >- Method reference verified against the entry's own PATHWAY line and against PMID:8385667, which shows the pathway runs in the dark in this organism. - id: PMID:8158642 title: Directed mutational analysis of bacteriochlorophyll a biosynthesis in Rhodobacter capsulatus. findings: - statement: >- A systematic directed mutational analysis of twelve open reading frames of the R. capsulatus 46 kb photosynthesis gene cluster. Mutations in seven of them abolished bacteriochlorophyll biosynthesis and caused accumulation of specific Mg-tetrapyrrole intermediates; four had no discernible pigment phenotype and one was partially disrupted. The cached record is abstract-only, and the abstract names no individual open reading frame. supporting_text: >- we have undertaken a systematic directed mutational analysis of 12 open reading frames in the photosynthesis gene cluster to evaluate whether individual open reading frames have a role in photopigment biosynthesis reference_section_type: ABSTRACT - statement: >- That bchF is among the open reading frames mutated, and that its disruption causes accumulation of chlorophyllide a and 3-vinyl bacteriochlorophyllide a, is established indirectly: a 2023 study cites this paper (DOI 10.1006/jmbi.1994.1260, reference 3 of its Crossref reference list) as a source for the R. capsulatus bchF deletion phenotype. supporting_text: >- mutations constructed in seven open reading frames resulted in a loss of bacteriochlorophyll biosynthesis, concomitant with the accumulation of specific intermediates in the Mg-tetrapyrrole biosynthetic pathway reference_section_type: ABSTRACT full_text_unavailable: true reference_review: relevance: HIGH correctness: VERIFIED review_notes: >- PubMed-verified (Bollivar, Suzuki, Beatty, Dobrowolski & Bauer, J Mol Biol 237:622-640, 1994). This is a cluster-wide mutational analysis of the R. capsulatus photosynthesis gene cluster, so its use by several bch gene annotations from one paper is appropriate rather than over-harvesting. The cached record is abstract-only, so the identity of the individual mutants cannot be read directly; PMID:36971575 cites this DOI for the R. capsulatus bchF deletion phenotype, which corroborates that bchF was among them. - id: PMID:8385667 title: bchFNBH bacteriochlorophyll synthesis genes of Rhodobacter capsulatus and identification of the third subunit of light-independent protochlorophyllide reductase in bacteria and plants. findings: - statement: >- This is the paper that defined the bchF locus. Pigments accumulated by strains carrying physically mapped transposon insertions were analysed spectroscopically and used to assign bchF, bchN, bchB and bchH, and bchF was identified as the bacteriochlorophyll a-specific enzyme that adds water across the vinyl group at the position now numbered C-3. supporting_text: >- bchF encodes a bacteriochlorophyll alpha-specific enzyme that adds water across the 2-vinyl group. reference_section_type: ABSTRACT - statement: >- The pathway bchF serves runs in the dark in this organism: protochlorophyllide reduction is carried out by BchN, BchB and BchL rather than by a light-dependent reductase. supporting_text: bchN and bchB are required for protochlorophyllide reduction in the dark (along with bchL) reference_section_type: ABSTRACT full_text_unavailable: true reference_review: relevance: HIGH correctness: VERIFIED review_notes: >- PubMed-verified; it is reference [1] of the UniProt entry for P26165 and the source of the gene's name and sequence. It is the strongest R. capsulatus-specific evidence for bchF function and is cited by no GOA row for this protein. - id: PMID:36971575 title: The Photoactive Photosynthetic Reaction Center of a Rhodobacter sphaeroides Mutant Lacking 3-Vinyl (Bacterio)Chlorophyllide a Hydratase Contains 3-Vinyl Bacteriochlorophyll a. findings: - statement: >- Places BchF in the bacteriochlorophyll a pathway as the 3-vinyl (bacterio)chlorophyllide a hydratase, acting either on 3-vinyl bacteriochlorophyllide a after chlorophyllide a reductase or on chlorophyllide a before it, with BchC oxidising the resulting 3-(1-hydroxyethyl) group to the 3-acetyl group of bacteriochlorophyllide a. supporting_text: >- followed by hydration of the 3V group to the 3-hydroxyethyl (3HE) group of 3HE-Bchlide a by 3-vinyl (bacterio)chlorophyllide a [3V-(B)Chlide a] hydratase (BchF) reference_section_type: INTRODUCTION - statement: >- States the bchF deletion phenotype for both Rhodobacter species, citing (as its references 2-4) Bollivar et al. 1994 for the R. capsulatus result. supporting_text: >- Deletion of bchF in Rhodobacter sphaeroides and Rhodobacter capsulatus results in the accumulation of Chlide a and 3V-Bchlide a reference_section_type: INTRODUCTION reference_review: relevance: HIGH correctness: VERIFIED review_notes: >- Full text cached. Primary data are on R. sphaeroides, but the introduction states the R. capsulatus bchF phenotype and cites PMID:8158642 for it; the citation was checked against the Crossref reference list of 10.1128/spectrum.03878-22, whose reference 3 is 10.1006/jmbi.1994.1260. - id: PMID:36822492 title: 'In vitro reversible dehydration in C3-substituents of zinc chlorophyll analogs by BchF and BchV enzymes: Stereoselectivity and substrate specificity in the dehydration.' findings: - statement: >- Direct enzymology on the BchF family: recombinant BchF (and its paralogue BchV) from Chlorobaculum tepidum, expressed in E. coli, catalyses hydration of the C3-vinyl group to a C3-1-hydroxyethyl group and runs the reaction in reverse, establishing the family as a stereoselective hydratase/dehydratase. supporting_text: BchF and BchV enzymes catalyze the hydration of a C3-vinyl to C3-1-hydroxyethyl group reference_section_type: ABSTRACT - statement: >- The assays were done on proteins from Chlorobaculum tepidum, not from Rhodobacter capsulatus, so this is orthologue-level rather than species-level evidence. supporting_text: obtained from extracts of Escherichia coli overexpressing the respective genes from Chlorobaculum tepidum reference_section_type: ABSTRACT full_text_unavailable: true reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: >- PubMed-verified. Relevant as family-level enzymological grounding for the hydratase activity; the protein assayed is the C. tepidum orthologue. - id: PMID:28643169 title: In vitro enzymatic assays of photosynthetic bacterial 3-vinyl hydratases for bacteriochlorophyll biosyntheses. findings: - statement: >- Paralogous 3-vinyl hydratases from Chlorobaculum tepidum, Chlorobaculum limnaeum, Chloroflexus aurantiacus and Chloracidobacterium thermophilum were all shown to be active hydratases in vitro, and the same 3-vinyl hydration step is identified as part of bacteriochlorophyll a biosynthesis. supporting_text: All the hydratases showed hydration activities. reference_section_type: ABSTRACT - statement: >- The 3-vinyl hydration carried out by this enzyme family precedes the oxidation that installs the 3-acetyl group of bacteriochlorophyll a. supporting_text: >- This 3-vinyl hydration also occurs in BChl a biosynthesis, followed by oxidation that introduces an acetyl group at the 3-position. reference_section_type: ABSTRACT full_text_unavailable: true reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: >- PubMed-verified. Breadth evidence that the 3-vinyl hydratase family is catalytically active across chlorophototroph lineages. - id: file:genes/RHOCB/bchF/bchF-uniprot.txt title: UniProt record for Rhodobacter capsulatus SB1003 bchF (P26165) findings: - statement: >- The entry assigns P26165 to the BchF family by three independent signatures (NCBIfam TIGR02020, Pfam PF07284, InterPro IPR009905) and places it in the light-independent bacteriochlorophyll biosynthesis pathway. supporting_text: 'NCBIfam; TIGR02020; BchF; 1.' record_section: - DR:NCBIfam - statement: >- Protein existence is at the prediction level: no experiment on the P26165 protein itself is recorded, and the entry carries no CC FUNCTION, no catalytic activity, and no transmembrane features despite the sequence being strongly hydrophobic. supporting_text: 'PE 4: Predicted;' record_section: - PE existing_annotations: - term: id: GO:0016836 label: hydro-lyase activity evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR009905 review: summary: >- Right enzyme class, but at the top of the hydro-lyase hierarchy. The reaction the BchF family performs is specifically hydration of the C-3 vinyl group of (bacterio)chlorophyllide a (EC 4.2.1.165), and GO has no term for it, so the replacement has to be a new term request. action: MODIFY reason: >- The IEA rests on family membership, which is well supported here by three independent signatures (NCBIfam TIGR02020 BchF, Pfam PF07284, InterPro IPR009905), and the chemistry of the family is established: recombinant BchF from Chlorobaculum tepidum hydrates a C3-vinyl group to a C3-1-hydroxyethyl group in vitro and runs the reaction reversibly (PMID:36822492), and the same activity was confirmed for 3-vinyl hydratases from four chlorophototroph lineages (PMID:28643169). For R. capsulatus itself, transposon-insertion mutants identified bchF as the enzyme that adds water across the vinyl group (PMID:8385667). So the hydro-lyase claim is true but far less specific than the evidence supports. No is_a child of GO:0016836 fits: a QuickGO search for "bacteriochlorophyllide" returns only GO:0036354, which is the activity of the NEXT enzyme (BchC, the 3-(1-hydroxyethyl) dehydrogenase), and no hydratase term for this reaction exists (checked 2026-11-01). The replacement is therefore the NTR filed in proposed_new_terms. Note the term is right on the chemistry and is not a neighbouring step's term: the preceding step is a reduction by BchXYZ and the following step an NAD-dependent oxidation by BchC, neither of them a hydro-lyase. proposed_replacement_terms: - id: NTR label: 3-vinyl bacteriochlorophyllide a hydratase activity supported_by: - reference_id: PMID:8385667 supporting_text: >- bchF encodes a bacteriochlorophyll alpha-specific enzyme that adds water across the 2-vinyl group. reference_section_type: ABSTRACT - reference_id: PMID:36822492 supporting_text: >- The results indicated that both BchF and BchV did function as a hydratase/dehydratase and could play a role in the C31-epimerization. reference_section_type: ABSTRACT - reference_id: file:genes/RHOCB/bchF/bchF-uniprot.txt supporting_text: 'Pfam; PF07284; BCHF; 1.' record_section: - DR:Pfam - term: id: GO:0019685 label: photosynthesis, dark reaction evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: involved_in supporting_entities: - InterPro:IPR009905 review: summary: >- Wrong term, reached through a homonym. GO:0019685 "photosynthesis, dark reaction" is the carbon-fixation grouping term, defined as the reduction of carbon dioxide to sugar, and bchF has no role in carbon fixation. The InterPro2GO rule evidently read the family's "light-independent" pathway as GO's "dark reaction". action: REMOVE reason: >- The GO definition of GO:0019685 is "A complex cycle of enzyme-mediated reactions which catalyzes the reduction of carbon dioxide to sugar... The reduced NADP (NADPH) and ATP are produced by the 'light' reactions" (QuickGO, 2026-11-01). BchF hydrates the C-3 vinyl group of a Mg-tetrapyrrole (PMID:36971575, PMID:8385667); it neither fixes carbon nor participates in a cycle that does. This is a demonstrably wrong electronic mapping rather than an over-propagated but defensible inference, which is the sanctioned ground for REMOVE on an IEA row. The cause is visible in the InterPro entry's own text, which describes IPR009905 as involved in the light-independent bacteriochlorophyll biosynthesis pathway, and the error is systematic: all four InterPro2GO rules that emit GO:0019685 are light-independent (bacterio)chlorophyll-synthesis families (IPR005969, IPR005970, IPR005971, IPR009905), and QuickGO reports 3,731 annotations to GO:0019685 of which all 3,731 cite GO_REF:0000002 - i.e. every GOA use of this Calvin-cycle term descends from this one misreading. The correct term for this family is GO:0036070, which the gene already carries from UniPathway, so nothing is lost by removing the row. supported_by: - reference_id: PMID:36971575 supporting_text: >- followed by hydration of the 3V group to the 3-hydroxyethyl (3HE) group of 3HE-Bchlide a by 3-vinyl (bacterio)chlorophyllide a [3V-(B)Chlide a] hydratase (BchF) reference_section_type: INTRODUCTION - reference_id: file:genes/RHOCB/bchF/bchF-uniprot.txt supporting_text: 'InterPro; IPR009905; BCHF.' record_section: - DR:InterPro source_defects: - defect_type: WRONG_TERM resource: INTERPRO record: InterPro:IPR009905 slot: interpro2go mapping requested_change: DELETE_VALUE asserted_value: GO:0019685 cause: LABEL_WITHOUT_DEFINITION confidence: CONFIRMED explanation: >- InterPro2GO is the resource behind GO_REF:0000002. The interpro2go file maps IPR009905 to GO:0019685 "photosynthesis, dark reaction". That term is the carbon-fixation grouping term, its is_a children being GO:0019253 reductive pentose-phosphate cycle, GO:0009760 C4 photosynthesis and GO:0009761 CAM photosynthesis, and its definition describes the reduction of CO2 to sugar. The mapping is explicable only as reading the label "dark reaction" as "light-independent", which is what the entry names say. The request is deletion rather than a repoint because IPR009905 already carries GO:0030494 bacteriochlorophyll biosynthetic process beside the wrong value, so removing GO:0019685 loses nothing; GO:0036070 would be a further refinement, not a replacement. The same deletion applies to the three light-independent protochlorophyllide reductase entries IPR005969, IPR005970 and IPR005971, which already carry GO:0015995 chlorophyll biosynthetic process and GO:0016730. Those three span both pigments, so no single BP mapping is precise for them (GO:0036068 light-independent chlorophyll biosynthetic process for ChlB/N/L against GO:0036070 for BchB/N/L); GO:0016630 protochlorophyllide reductase activity would be exact for all three. Because these four entries are the only interpro2go uses of GO:0019685, and QuickGO reports that all 3,731 GOA annotations to GO:0019685 are IEA from GO_REF:0000002 assigned by InterPro, the deletions remove every current GOA use of the term. Written up in projects/CACAO/upstream-interpro2go-dark-reaction.md, which also asks GO separately to rename the term and generalise its definition. supported_by: - reference_id: PMID:36971575 supporting_text: >- oxidation of the 3HE group by 3HE-Bchlide a dehydrogenase (BchC) to form the acetyl group of Bchlide a, which is a direct precursor of Bchl a reference_section_type: INTRODUCTION - reference_id: file:genes/RHOCB/bchF/bchF-uniprot.txt supporting_text: >- PATHWAY: Porphyrin-containing compound metabolism; bacteriochlorophyll biosynthesis (light-independent). record_section: - CC:PATHWAY - term: id: GO:0030494 label: bacteriochlorophyll biosynthetic process evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: involved_in supporting_entities: - InterPro:IPR009905 review: summary: >- True, and the right pathway, but one level less specific than the gene's own evidence supports. The bacteriochlorophyll a pathway of R. capsulatus is light-independent, so GO:0036070 - which the gene already carries from UniPathway - is the supported term. action: MODIFY reason: >- The family-level BP inference is sound: IPR009905 members act in bacteriochlorophyll biosynthesis, and in R. capsulatus the hydration step was genetically assigned to bchF (PMID:8385667). The refinement is warranted because protochlorophyllide reduction in this organism is carried out in the dark by BchL, BchN and BchB (PMID:8385667), making the whole branch light-independent; GO:0036070 is an is_a descendant of GO:0030494 and is already asserted on this gene from UniPathway UPA00671, so the replacement is a term the gene carries rather than a new claim. Keeping the parent as well would only restate it. proposed_replacement_terms: - id: GO:0036070 label: light-independent bacteriochlorophyll biosynthetic process supported_by: - reference_id: PMID:8385667 supporting_text: bchN and bchB are required for protochlorophyllide reduction in the dark (along with bchL) reference_section_type: ABSTRACT - reference_id: file:genes/RHOCB/bchF/bchF-uniprot.txt supporting_text: >- PATHWAY: Porphyrin-containing compound metabolism; bacteriochlorophyll biosynthesis (light-independent). record_section: - CC:PATHWAY - term: id: GO:0030494 label: bacteriochlorophyll biosynthetic process evidence_type: IMP original_reference_id: PMID:8158642 qualifier: involved_in review: summary: >- CACAO row. The cited paper is a genuine, cluster-wide mutational analysis of the R. capsulatus photosynthesis gene cluster and the claim it is used for is correct, so the row stands; it is refined to the light-independent child term for the same reason as the IEA row carrying this term. action: MODIFY reason: >- What the paper shows for this gene: PMID:8158642 constructed directed mutations in twelve open reading frames of the 46 kb R. capsulatus photosynthesis gene cluster, of which seven lost bacteriochlorophyll biosynthesis with accumulation of specific Mg-tetrapyrrole intermediates. The cached record is abstract-only (full_text_available: false) and the abstract names no individual open reading frame, so bchF's inclusion cannot be read off it; it is corroborated independently, because PMID:36971575 states that deletion of bchF in R. sphaeroides and R. capsulatus causes accumulation of chlorophyllide a and 3-vinyl bacteriochlorophyllide a and cites this paper's DOI (10.1006/jmbi.1994.1260, reference 3 of its Crossref reference list) among its sources for that statement. That phenotype - loss of bacteriochlorophyll with accumulation of the unhydrated precursor - is exactly what an IMP for bacteriochlorophyll biosynthesis on a 3-vinyl hydratase should rest on, and it agrees with the independent transposon-mutant assignment of bchF in PMID:8385667. The term matches the claim; it is simply one level general, and GO:0036070 is supported because the pathway runs in the dark in this organism. GONUTS could not be consulted (see below), so the judgement is made from the literature; per the curator-deference rule no REMOVE was considered on an abstract alone, and none is warranted. On the batch-3 screen that selected this gene: the four CACAO rows citing PMID:8158642 span NCBITaxon:272942 (bchF, bchE, bchJ, all _RHOCB entries on the SB1003 genome) and NCBITaxon:1061 (bchM, the legacy species-level BCHM_RHOCA entry). Both ids denote Rhodobacter capsulatus, 1061 is the species node above the 272942 strain node, and the paper worked in R. capsulatus, so no isolate boundary is crossed and no locus mismatch exists; the split is UniProt entry granularity, not an annotation defect, and nothing is reportable against CACAO. GONUTS (gowiki.tamu.edu) was retried on 2026-11-01 and now returns HTTP 410 Gone with the notice "GONUTS has been retired"; the Wayback availability API has no snapshot of the relevant protein or paper pages, so that route is permanently closed rather than temporarily offline. proposed_replacement_terms: - id: GO:0036070 label: light-independent bacteriochlorophyll biosynthetic process additional_reference_ids: - PMID:36971575 - PMID:8385667 supported_by: - reference_id: PMID:8158642 supporting_text: >- mutations constructed in seven open reading frames resulted in a loss of bacteriochlorophyll biosynthesis, concomitant with the accumulation of specific intermediates in the Mg-tetrapyrrole biosynthetic pathway reference_section_type: ABSTRACT - reference_id: PMID:36971575 supporting_text: >- Deletion of bchF in Rhodobacter sphaeroides and Rhodobacter capsulatus results in the accumulation of Chlide a and 3V-Bchlide a reference_section_type: INTRODUCTION - reference_id: PMID:8385667 supporting_text: >- The pigments accumulated in strains with physically mapped transposon insertion mutations are analyzed by absorbance and fluorescence spectroscopy, allowing us to assign the genes as bchF, bchN, bchB, and bchH, in that order. reference_section_type: ABSTRACT - term: id: GO:0036070 label: light-independent bacteriochlorophyll biosynthetic process evidence_type: IEA original_reference_id: GO_REF:0000041 qualifier: involved_in supporting_entities: - UniPathway:UPA00671 review: summary: >- Correct and appropriately specific: this is the core process for bchF, and the pathway's light-independence is established genetically in this organism rather than merely assumed from the UniPathway label. action: ACCEPT reason: >- bchF performs a dedicated step of the bacteriochlorophyll a-specific branch - hydration of the C-3 vinyl group, with BchC oxidising the product to the 3-acetyl group of bacteriochlorophyllide a (PMID:36971575) - and its disruption abolishes bacteriochlorophyll synthesis with accumulation of the unhydrated intermediate (PMID:8158642, PMID:36971575). The "light-independent" qualifier is not taken from the UniPathway string alone: in R. capsulatus protochlorophyllide reduction is carried out in the dark by BchL, BchN and BchB (PMID:8385667), so the branch bchF belongs to operates without light. No more specific descendant exists, so this is as precise as the ontology allows. supported_by: - reference_id: PMID:36971575 supporting_text: >- followed by hydration of the 3V group to the 3-hydroxyethyl (3HE) group of 3HE-Bchlide a by 3-vinyl (bacterio)chlorophyllide a [3V-(B)Chlide a] hydratase (BchF) reference_section_type: INTRODUCTION - reference_id: PMID:8385667 supporting_text: bchN and bchB are required for protochlorophyllide reduction in the dark (along with bchL) reference_section_type: ABSTRACT core_functions: - description: >- Hydration of the C-3 vinyl group of chlorophyllide a or 3-vinyl bacteriochlorophyllide a to the corresponding 3-(1-hydroxyethyl) compound, the committed step that allows BchC to install the 3-acetyl group of bacteriochlorophyllide a, within the light-independent bacteriochlorophyll a biosynthetic pathway of Rhodobacter capsulatus. proposed_molecular_function: 3-vinyl bacteriochlorophyllide a hydratase activity directly_involved_in: - id: GO:0036070 label: light-independent bacteriochlorophyll biosynthetic process supported_by: - reference_id: PMID:8385667 supporting_text: >- bchF encodes a bacteriochlorophyll alpha-specific enzyme that adds water across the 2-vinyl group. reference_section_type: ABSTRACT - reference_id: PMID:36971575 supporting_text: >- followed by hydration of the 3V group to the 3-hydroxyethyl (3HE) group of 3HE-Bchlide a by 3-vinyl (bacterio)chlorophyllide a [3V-(B)Chlide a] hydratase (BchF) reference_section_type: INTRODUCTION - reference_id: PMID:36822492 supporting_text: BchF and BchV enzymes catalyze the hydration of a C3-vinyl to C3-1-hydroxyethyl group reference_section_type: ABSTRACT - reference_id: PMID:8158642 supporting_text: >- mutations constructed in seven open reading frames resulted in a loss of bacteriochlorophyll biosynthesis, concomitant with the accumulation of specific intermediates in the Mg-tetrapyrrole biosynthetic pathway reference_section_type: ABSTRACT knowledge_gaps: - gap_statement: >- Which substrate BchF actually hydrates in vivo - chlorophyllide a before chlorophyllide a reductase (BchXYZ) acts, or 3-vinyl bacteriochlorophyllide a after it - is undetermined, and so is whether the two routes are used in parallel or one is preferred. boundary: >- The chemistry is settled (hydration of the C-3 vinyl group to a 3-(1-hydroxyethyl) group, shown in vitro for family members and genetically in R. capsulatus), and the product is settled (the substrate of BchC). What is not settled is the order of the hydration and the C-7/C-8 reduction in the cell. gap_kind: - BIOLOGY dark_aspect: MF_DARK status: OPEN significance: >- The branch order determines which intermediate accumulates in each mutant and whether BchF and BchXYZ channel substrate between them; it also decides whether the enzyme's physiological substrate is a chlorin or a bacteriochlorin, which bears on the stereochemistry of the product. resolution: >- Steady-state kinetics of purified R. capsulatus BchF on chlorophyllide a versus 3-vinyl bacteriochlorophyllide a, combined with pulse labelling of intermediates in wild-type and bchXYZ/bchF mutant backgrounds. provenance: - reference_id: PMID:36971575 supporting_text: Alternatively, Chlide a may be metabolized by BchF to yield 3HE-Chlide a reference_section_type: INTRODUCTION - reference_id: PMID:36971575 supporting_text: which resulted in cellular accumulation of Chlide a and 3V-Bchlide a reference_section_type: OTHER proposed_new_terms: - proposed_name: 3-vinyl bacteriochlorophyllide a hydratase activity proposed_definition: >- Catalysis of the reaction: 3-vinyl bacteriochlorophyllide a + H2O = 3-(1-hydroxyethyl)bacteriochlorophyllide a. The enzyme also acts on chlorophyllide a to give 3-(1-hydroxyethyl)chlorophyllide a, and the reaction is reversible. justification: >- GO has no term for the reaction catalysed by the BchF family (EC 4.2.1.165), even though the term for the next step of the same pathway exists (GO:0036354 bacteriochlorophyllide-a dehydrogenase activity, the BchC activity). A QuickGO search for "bacteriochlorophyllide" returns only GO:0036354, and GO:0016836 hydro-lyase activity has no child covering this reaction (checked 2026-11-01). As a result every BchF protein in GOA sits at the generic hydro-lyase parent. Suggested mappings: EC 4.2.1.165, MetaCyc MONOMER-13252, BRENDA 4.2.1.165. proposed_parent: id: GO:0016836 label: hydro-lyase activity supported_by: - reference_id: PMID:36822492 supporting_text: BchF and BchV enzymes catalyze the hydration of a C3-vinyl to C3-1-hydroxyethyl group reference_section_type: ABSTRACT - reference_id: PMID:36822492 supporting_text: >- The results indicated that both BchF and BchV did function as a hydratase/dehydratase and could play a role in the C31-epimerization. reference_section_type: ABSTRACT - reference_id: PMID:28643169 supporting_text: >- Biosynthesis of chlorosomal pigments involves stereoselective conversion of 3-vinyl group to 3-(1-hydroxyethyl) group facilitated by a 3-vinyl hydratase. reference_section_type: ABSTRACT - reference_id: PMID:8385667 supporting_text: >- bchF encodes a bacteriochlorophyll alpha-specific enzyme that adds water across the 2-vinyl group. reference_section_type: ABSTRACT suggested_questions: - question: >- Is R. capsulatus BchF an integral membrane protein, and if so what is the topology and which side of the membrane holds the active site? The sequence is strongly hydrophobic over roughly residues 30-165, BchF is usually discussed as membrane-bound, yet UniProt asserts no transmembrane feature for P26165 and GOA carries no cellular-component annotation at all. experts: - Jiro Harada - Hitoshi Tamiaki - question: >- In the cell, does BchF act on chlorophyllide a before chlorophyllide a reductase (BchXYZ) or on 3-vinyl bacteriochlorophyllide a after it, and do the two enzymes channel substrate between them? experts: - Jiro Harada - Yusuke Tsukatani - question: >- Given that BchF runs the hydration reversibly in vitro, is the reverse (dehydratase) direction physiologically relevant in a purple bacterium, for example during bacteriochlorophyll turnover or under oxidative stress? experts: - Hitoshi Tamiaki suggested_experiments: - hypothesis: >- R. capsulatus BchF is a catalytically autonomous 3-vinyl hydratase whose preferred substrate determines the order of the hydration and C-7/C-8 reduction steps in vivo. description: >- Express R. capsulatus bchF in E. coli, as was done for the Chlorobaculum tepidum orthologue, and assay the detergent-solubilised enzyme on chlorophyllide a and on 3-vinyl bacteriochlorophyllide a, measuring steady-state kinetics and the stereochemistry of the 3-(1-hydroxyethyl) product by chiral HPLC. This would give the first direct enzymology on the R. capsulatus protein, whose activity is at present inferred from mutant pigment phenotypes and orthologue assays, and would settle the substrate preference. experiment_type: heterologous expression and in vitro enzyme assay - hypothesis: >- BchF is an integral membrane protein of the cytoplasmic membrane, so its reaction occurs at the membrane where the downstream enzymes BchC, BchG and BchP also act. description: >- Determine the subcellular distribution and topology of BchF in R. capsulatus by fractionating cells into soluble and membrane fractions and immunoblotting for an epitope-tagged BchF, then map the topology by cysteine-accessibility (substituted cysteine scanning with a membrane-impermeant thiol reagent) or by dual PhoA/LacZ reporter fusions at successive positions. A positive result would let a cellular-component annotation be made for a protein that currently has none. experiment_type: subcellular fractionation and membrane topology mapping - hypothesis: >- bchF of R. capsulatus is functionally interchangeable with the 3-vinyl hydratases of distant chlorophototrophs. description: >- Complement a defined R. capsulatus bchF deletion with bchF or bchV from Chlorobaculum tepidum, Chloroflexus aurantiacus and Chloracidobacterium thermophilum, and score restoration of bacteriochlorophyll a and of photoheterotrophic growth together with the pigment profile by HPLC. This tests whether the in vitro activities reported for those orthologues correspond to the physiological step in a purple bacterium, and whether any of them instead yields the epimeric product. experiment_type: cross-species genetic complementation with pigment analysis