bchE encodes the anaerobic magnesium-protoporphyrin IX monomethyl ester (MPE) cyclase of Rhodobacter capsulatus, the enzyme that closes the isocyclic fifth (E) ring of the bacteriochlorophyll macrocycle. It converts Mg-protoporphyrin IX 13-monomethyl ester to 3,8-divinyl protochlorophyllide a by a six-electron oxidation that installs an oxo group at C13(1), drawing that oxygen from water rather than from molecular oxygen (EC 1.21.98.3). BchE is a cobalamin-dependent radical S-adenosyl-L-methionine enzyme: an N-terminal B12-binding domain carries adenosylcobalamin and a radical SAM core carries a [4Fe-4S] cluster on the canonical CxxxCxxC motif (Cys204, Cys208, Cys211), from which reductive cleavage of SAM generates the 5'-deoxyadenosyl radical that abstracts the first hydrogen from the substrate's 13(1) position. The same net conversion is performed in oxygenic phototrophs and in aerobically growing anoxygenic phototrophs by the unrelated di-iron oxygenase AcsF, which uses O2 and NADPH; R. capsulatus has no AcsF, so BchE is the organism's only route from MPE to protochlorophyllide and loss of bchE blocks bacteriochlorophyll a synthesis with stoichiometric accumulation of MPE. The enzyme acts in the light-independent (dark-operative) branch of chlorophyll/bacteriochlorophyll biosynthesis and is active only under anaerobic, photosynthetic growth conditions; its native localization is membrane-associated and it requires an additional small soluble host component for activity.
Definition: Catalysis of the reaction: Mg-protoporphyrin IX 13-monomethyl ester + 3 S-adenosyl-L-methionine + H2O = 3,8-divinyl protochlorophyllide a + 3 5'-deoxyadenosine + 3 L-methionine + 4 H(+). This oxygen-independent, cobalamin- and radical-S-adenosyl-L-methionine-dependent six-electron oxidation installs the oxo group at C13(1) and closes the isocyclic (E) ring, taking the oxo oxygen from water rather than from molecular oxygen.
Justification: GO has no term for EC 1.21.98.3 / RHEA:49096, the anaerobic route to protochlorophyllide catalysed by BchE. The only existing MPE cyclase term, GO:0048529, is defined on the aerobic di-iron oxygenase reaction of AcsF (EC 1.14.13.81, RHEA:33235) and is_a GO:0016709, so it cannot describe BchE without asserting O2 and NADPH chemistry that BchE does not perform - yet GO:0048529 is in fact propagated to bchE orthologs by IEA, which is a recurring misassignment of this step. Bacteria and archaea that make bacteriochlorophyll anaerobically (Rhodobacter, Rubrivivax, Chlorobium, Heliobacillus) need the distinct activity, and R. capsulatus, which has no AcsF at all, is the clearest case. Without the term, the enzyme's only molecular-function annotation is the ontology root GO:0003824.
Parent term: oxidoreductase activity, acting on X-H and Y-H to form an X-Y bond
Mappings:
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MODIFY | Summary: BchE is unquestionably an enzyme, and the two InterPro signatures behind this row (Elp3/MiaA/NifB-like rSAM, IPR006638; rSAM, IPR007197) correctly identify its radical-SAM core. But GO:0003824 is the root of the molecular function ontology's catalytic branch and says nothing about what BchE does. It is standing in for a term GO does not yet have. Reason: BchE's reaction is EC 1.21.98.3 / RHEA:49096, a cobalamin- and radical-SAM-dependent six-electron oxidation of Mg-protoporphyrin IX 13-monomethyl ester to 3,8-divinyl protochlorophyllide a that takes its oxo oxygen from water. GO has no term for it. The only MPE cyclase term, GO:0048529 "magnesium-protoporphyrin IX monomethyl ester (oxidative) cyclase activity", is defined on the AEROBIC reaction (EC 1.14.13.81, RHEA:33235, "+ 3 NADPH + 3 H+ + 3 O2 = divinylprotochlorophyllide + 3 NADP+ + 5 H2O") and is_a GO:0016709 (incorporation of molecular oxygen) - the chemistry of AcsF, not of BchE. Annotating BchE to GO:0048529 would be exactly the historical misassignment of this step, and P26168 commendably does not carry it; QuickGO ontology searches for "1.21.98.3", "RHEA:49096" and "anaerobic magnesium-protoporphyrin" return no matching term. The replacement is therefore the new term proposed in this review, parented under GO:0046992 (the EC 1.21 class). The underlying activity is not in doubt: it is UniProt's curated CATALYTIC ACTIVITY with ECO:0000269, validated in vitro for the R. capsulatus enzyme by H2(18)O label transfer onto the C13(1)-oxo group [PMID:33211085] and reconstituted with purified protein for a BchE ortholog [PMID:42748141]. Proposed replacements: anaerobic magnesium-protoporphyrin IX monomethyl ester cyclase activity Supporting Evidence: file:genes/RHOCB/bchE/bchE-uniprot.txt Reaction=Mg-protoporphyrin IX 13-monomethyl ester + 3 S-adenosyl-L-methionine + H2O = 3,8-divinyl protochlorophyllide a + 3 5'-deoxyadenosine + 3 L-methionine + 4 H(+) PMID:33211085 We established a spectroscopic in vitro activity assay which was subsequently validated by HPLC analyses and H218O isotope label transfer onto the carbonyl-group (C-131-oxo) of the isocyclic ring of Pchlide. file:genes/RHOCB/bchE/bchE-uniprot.txt Pfam; PF04055; Radical_SAM; 1. |
| GO:0005829 cytosol | IEA GO_REF:0000118 | MARK AS OVER ANNOTATED | Summary: A TreeGrafter node-level location, not an observation on BchE. Direct subcellular fractionation of R. capsulatus places BchE on the membrane and identifies a separate small soluble component as the cytosolic requirement for activity, so asserting the cytosol as BchE's location overstates what is known. Reason: P26168 has no transmembrane segment and no signal peptide in UniProt, which is precisely the feature profile that makes a tree-grafted cytosol default the expected machine output; the row therefore carries no independent information about this protein. Against it stands direct experimental fractionation in the native host: Wiesselmann et al. report "membrane-localized BchE" requiring a distinct heat-sensitive cytosolic component, later narrowed by size fractionation to a 3-10 kDa species [PMID:33211085]. Consistent with a membrane-associated enzyme, BchE has a long history of insolubility on recombinant expression, and even when soluble R. capsulatus BchE was finally obtained it was catalytically dead [PMID:42748141]. This is MARK_AS_OVER_ANNOTATED rather than REMOVE because the fractionation evidence is available to us only as an abstract and the enzyme may well partition between a membrane-bound active pool and a soluble pool; it is not MODIFY to a membrane term, because asserting a specific membrane location from one abstract would substitute one unsupported location for another. Recorded as a knowledge gap and a suggested experiment instead. Propagation Review Root cause: SOURCE WEAK OR INFERRED Failure modes: COMPARTMENT OR COMPLEX MISMATCH Sources checked: PANTHER:PTN000228879 SOURCE WEAK OR INFERRED Ancestral node location for the BchE-like family; no sub-cellular assay on any member grounds it, and the one direct fractionation of a member (R. capsulatus BchE) found the protein membrane-associated. Supporting Evidence: PMID:33211085 Subcellular fractionation experiments revealed that membrane-localized BchE requires an additional, heat-sensitive cytosolic component for activity. PMID:33211085 Size-fractionation of the soluble R. capsulatus fraction indicated that enzymatic activity relies on a specific component with an estimated molecular mass between 3 and 10 kDa. PMID:42748141 detailed mechanistic analysis has been impeded by the inability to obtain soluble, catalytically active enzyme |
| GO:0030494 bacteriochlorophyll biosynthetic process | IMP PMID:8158642 Directed mutational analysis of bacteriochlorophyll a biosyn... | MODIFY | Summary: The CACAO row. Bollivar et al. 1994 is a cluster-wide directed mutational analysis of 12 photosynthesis-gene-cluster reading frames in which seven disruptions abolished bacteriochlorophyll synthesis with accumulation of specific Mg-tetrapyrrole intermediates; for bchE that intermediate is MPE. The claim is correct and the evidence code is right. The entry already carries the more specific GO:0036070, of which GO:0030494 is an ancestor, so the row is refined rather than challenged. Reason: This is a sound student annotation, and the strain/locus questions that this gene was screened for do not hold up: the paper is a systematic survey of the whole gene cluster, so the fact that bchE, bchF and bchJ all cite it is what that experiment supports, not a sign that one locus's result was spread across its neighbours. The gene-specific lesion is independently corroborated - the MPE-accumulating bchE transposon mutant is reported by Yang and Bauer as ORF F1025 [PMID:2203738] and named as bchE by Gough et al. [PMID:10841582]. The only change warranted is granularity. R. capsulatus makes bacteriochlorophyll only under anaerobic, photosynthetic conditions and has no AcsF, so BchE is the obligate dark/oxygen-independent cyclase [PMID:42748141]; UniProt's curated pathway statement is explicitly "bacteriochlorophyll biosynthesis (light-independent)". QuickGO gives GO:0030494 among the is_a/part_of ancestors of GO:0036070, which this entry already carries by IEA, so MODIFY points this experimental row at the specific term it in fact supports rather than leaving the strongest evidence on the broader one. Proposed replacements: light-independent bacteriochlorophyll biosynthetic process Supporting Evidence: PMID:8158642 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 PMID:8158642 The results of this analysis demonstrate that 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. PMID:10841582 MPE accumulates in transposon mutants of the Rhodobacter capsulatus bchE gene, suggesting that the encoded protein is required for MPE-cyclase activity file:genes/RHOCB/bchE/bchE-uniprot.txt Porphyrin-containing compound metabolism; bacteriochlorophyll biosynthesis (light-independent). |
| GO:0031419 cobalamin binding | IEA GO_REF:0000002 | ACCEPT | Summary: Correct and, unusually for an InterPro2GO row, independently grounded well beyond family membership. BchE carries a B12-binding domain at residues 9-143, the cyclase reaction in R. capsulatus has an absolute cobalamin requirement shown in vivo, candidate cobalamin ligands were identified by site-directed mutagenesis of this very protein, and a purified BchE ortholog shows a cob(II)alamin EPR signal. Reason: The domain assignment itself is sound (Pfam PF02310 B12-binding, PROSITE PS51332, UniProt DOMAIN 9..143), so the row would be defensible on membership alone - but it does not have to rest there. Gough et al. showed with B12-auxotrophic bluE/bluB mutants and an in vivo cyclase assay under inhibited protein synthesis that the MPE cyclase reaction has an absolute demand for B12 or adenosylcobalamin, which UniProt records as an ECO:0000269 adenosylcob(III)alamin cofactor [PMID:10841582]. Wiesselmann et al. then identified candidate cobalamin ligands (Asp248, Glu249, Leu29, Thr71, Val97) by complementation assay of 24 site-directed R. capsulatus BchE mutants [PMID:33211085]. York et al. finally confirmed bound cobalamin spectroscopically on purified protein, though on the Rubrivivax pictus ortholog rather than the R. capsulatus enzyme, which is why that line is corroborating rather than primary here [PMID:42748141]. Core: cobalamin is not an incidental ligand but the cofactor that initiates the chemistry. Supporting Evidence: file:genes/RHOCB/bchE/bchE-uniprot.txt Name=adenosylcob(III)alamin; Xref=ChEBI:CHEBI:18408; Evidence={ECO:0000269|PubMed:10841582}; PMID:10841582 Fig. 5 C shows that mutant MPE cyclase activity has an absolute demand for exogenous B 12 or AdCob. This experiment shows that a cobalamin cofactor acts directly on the cyclase reaction. PMID:33211085 Potential ligands of the [4Fe-4S] cluster (Cys204, Cys208, Cys211), of SAM (Phe210, Glu308 and Lys320) and of the proposed cobalamin cofactor (Asp248, Glu249, Leu29, Thr71, Val97) were identified. file:genes/RHOCB/bchE/bchE-uniprot.txt Pfam; PF02310; B12-binding; 1. |
| GO:0036070 light-independent bacteriochlorophyll biosynthetic process | IEA GO_REF:0000041 | ACCEPT | Summary: The right process term at the right granularity, and the one this gene's experimental evidence actually supports. BchE is the oxygen-independent cyclase of the dark-operative bacteriochlorophyll route, and R. capsulatus lacks the aerobic alternative entirely. Reason: The UniPathway mapping lands on the specific term by the accident of being a pathway-level assignment, but it happens to be exactly right. UniProt's curated PATHWAY statement is "bacteriochlorophyll biosynthesis (light-independent)", and the FUNCTION statement specifies the anaerobic, water-derived-oxygen chemistry that distinguishes this branch. York et al. state plainly that R. capsulatus has no AcsF, so every molecule of protochlorophyllide in this organism comes from BchE [PMID:42748141]; Bollivar et al.'s bchE disruption accordingly abolishes bacteriochlorophyll synthesis under photosynthetic growth [PMID:8158642]. Core. Supporting Evidence: file:genes/RHOCB/bchE/bchE-uniprot.txt Porphyrin-containing compound metabolism; bacteriochlorophyll biosynthesis (light-independent). file:genes/RHOCB/bchE/bchE-uniprot.txt Catalyzes the anaerobic formation of the isocyclic ring (E-ring) in Mg-protoporphyrin monomethyl ester (MPE) to yield protochlorophyllide a (PChlide a) via a six-electron oxidation and the formation of an oxo group at position C13 using oxygen from a water molecule. PMID:42748141 R. capsulatus does not utilize AcsF, so all production of PChlide must originate through the action of BchE. |
| GO:0046872 metal ion binding | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: The one chemistry claim on this entry that rests on family membership alone and is not entailed by the specific cofactor terms. BchE's metals are the cobalt held inside a corrin ring and the irons held inside a [4Fe-4S] cluster; GO files both away from "metal ion binding" deliberately, and no discrete metal-ion site is reported for BchE. Reason: The row comes from the cobalamin-binding domain signatures (IPR006158, IPR036724) via InterPro2GO. It is not redundant with the specific terms, so TOO_GENERAL does not apply: QuickGO gives the is_a/part_of ancestors of GO:0051536 as GO:0051540 metal cluster binding, GO:0036094 and GO:0005488 - not GO:0046872 - and the ancestors of GO:0031419 as GO:0046906 tetrapyrrole binding, GO:0019842 vitamin binding, GO:0036094 and GO:0005488, again not GO:0046872. Neither specific term entails this one, and GO's placement encodes a real distinction: the cobalt is coordinated by the corrin macrocycle of a bound cofactor and the irons by cluster sulfurs, neither being a free metal ion recruited to a dedicated site. Nothing in the literature on BchE reports such a site; the substrate's own magnesium is part of the Mg-porphyrin, not an enzyme-bound ion, and the structure-guided mutagenesis of 24 residues found ligands only for the cluster, SAM and cobalamin [PMID:33211085]. Not REMOVE: cobalt and iron really are present in the holoenzyme, so the term is uninformative and over-reaching rather than false. Raised for InterPro as a suggested question. Supporting Evidence: PMID:33211085 Potential ligands of the [4Fe-4S] cluster (Cys204, Cys208, Cys211), of SAM (Phe210, Glu308 and Lys320) and of the proposed cobalamin cofactor (Asp248, Glu249, Leu29, Thr71, Val97) were identified. file:genes/RHOCB/bchE/bchE-uniprot.txt Name=[4Fe-4S] cluster; Xref=ChEBI:CHEBI:49883; Evidence={ECO:0000305|PubMed:10841582}; |
| GO:0051536 iron-sulfur cluster binding | IEA GO_REF:0000002 | ACCEPT | Summary: Supported by more than the radical-SAM signature that generated it. The canonical CxxxCxxC motif is present and intact in P26168 (Cys204-x3-Cys208-x2-Cys211), those three cysteines were tested by site-directed mutagenesis of R. capsulatus BchE and identified as the cluster ligands, and a purified BchE ortholog gives a [4Fe-4S] EPR spectrum. Reason: Worth separating the strands, because the cluster was for two decades an inference. Gough et al. only proposed it from a cysteine spacing resemblance to biotin synthase, which is why UniProt's cofactor and BINDING lines for the cluster still carry ECO:0000305 rather than ECO:0000269 [PMID:10841582]. The inference has since been tested on this protein: Wiesselmann et al. identified Cys204, Cys208 and Cys211 as the [4Fe-4S] ligands by in vivo complementation assay of 24 site-directed BchE mutants [PMID:33211085], and those positions sit in the canonical radical-SAM motif in the deposited sequence (residues 195-215 read VAIPNMARGCPFTCSFCSQWK, i.e. C204-x3-C208-x2-C211), so this is not a case of a motif asserted from memory. York et al. then confirmed a [Fe4S4] cluster spectroscopically on purified protein, on the Rubrivivax pictus ortholog [PMID:42748141]. Core: cluster reduction and reductive SAM cleavage are the first step of the catalytic cycle. Supporting Evidence: PMID:33211085 Potential ligands of the [4Fe-4S] cluster (Cys204, Cys208, Cys211), of SAM (Phe210, Glu308 and Lys320) and of the proposed cobalamin cofactor (Asp248, Glu249, Leu29, Thr71, Val97) were identified. PMID:42748141 The enzyme was purified anaerobically and characterized by UV-Vis and electron paramagnetic resonance spectroscopy to confirm the presence of Cbl and a [Fe4S4] cluster. file:genes/RHOCB/bchE/bchE-uniprot.txt BINDING 204 /ligand="[4Fe-4S] cluster" file:genes/RHOCB/bchE/bchE-uniprot.txt DOMAIN 190..417 /note="Radical SAM core" |
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Download this section (compressed HTML)Q: Should GO add a molecular-function term for EC 1.21.98.3 / RHEA:49096, the anaerobic cobalamin- and radical-SAM-dependent MPE cyclase, so that BchE proteins stop being annotated either to the ontology root GO:0003824 or, worse, to GO:0048529, whose definition commits them to the O2- and NADPH-dependent AcsF reaction they do not catalyse?
Suggested experts: Joern Moser, Dieter Jahn, Squire J. Booker
Q: Should InterPro2GO drop GO:0046872 metal ion binding from the cobalamin-binding domain signatures IPR006158 and IPR036724? GO places cobalamin binding under tetrapyrrole and vitamin binding, not under metal ion binding, because the cobalt is held inside a bound corrin cofactor rather than at a protein metal-ion site; the mapping adds an unentailed claim to every B12-binding protein.
Q: Where is catalytically competent BchE in the R. capsulatus cell, and what is the 3-10 kDa soluble component it requires? Purified R. capsulatus BchE is soluble but yields only the off-pathway 575 nm olefin, and RcFdxA does not rescue it, so the physiological reductant or partner is still unidentified.
Suggested experts: Joern Moser, Nicholas J. York
Q: Why do the R. capsulatus and R. gelatinosus BchE proteins stall at the off-pathway olefin in vitro while the Rubrivivax pictus ortholog turns over fully - a missing host factor, a lost cofactor loading step, or a genuine mechanistic difference between these enzymes?
Suggested experts: Squire J. Booker
Experiment: Fractionate anaerobically, photosynthetically grown R. capsulatus SB1003 into membrane and soluble fractions with explicit marker controls for each compartment, and locate BchE in parallel by immunoblot and by MPE-to-PChlide activity. Repeat with a chromosomally tagged bchE allele to exclude overexpression artefacts, and test salt and detergent extractability to distinguish peripheral association from integral insertion.
Hypothesis: Catalytically competent BchE is membrane-associated in photosynthetically grown R. capsulatus, and the cytosol annotation reflects only a soluble inactive pool.
Type: subcellular fractionation with marker-controlled immunoblot and activity assay
Experiment: Reconstitute purified R. capsulatus BchE with size-fractionated soluble extract from photosynthetically grown wild-type cells, following PChlide formation by HPLC and fluorescence, and purify the active fraction to identity by activity-guided chromatography and mass spectrometry. Counter-screen candidate small redox proteins, including RcFdxA and other ferredoxins and flavodoxins, individually and in combination.
Hypothesis: The 3-10 kDa soluble requirement of R. capsulatus BchE is the missing component that converts purified RcBchE from an olefin-forming dead end into a productive cyclase.
Type: activity-guided reconstitution and partner identification
Experiment: Express and purify wild-type and C204A, C208A and C211A R. capsulatus BchE anaerobically and compare iron and sulfide content, UV-visible spectra and EPR spectra before and after dithionite reduction, to convert the mutagenesis inference into direct spectroscopic assignment on this protein rather than on the Rubrivivax ortholog.
Hypothesis: Cys204, Cys208 and Cys211 ligate the single [4Fe-4S] cluster of R. capsulatus BchE, as inferred from complementation of site-directed mutants.
Type: anaerobic purification with iron/sulfide quantitation and EPR spectroscopy
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