id: P0ABG1 gene_symbol: cdsA product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:83333 label: Escherichia coli K-12 description: CdsA is the CDP-diacylglycerol synthase of Escherichia coli, a polytopic protein of the inner (cytoplasmic) membrane that activates phosphatidic acid with CTP to give CDP-diacylglycerol and pyrophosphate. This is the single committed step between phosphatidic acid, the end point of de novo fatty-acid esterification onto glycerol 3-phosphate, and the liponucleotide from which every bacterial phospholipid head group is built, so flux through phosphatidylglycerol, cardiolipin and phosphatidylethanolamine all pass through it. The enzyme works on membrane-embedded substrate and shows surface-dilution kinetics with a sequential rather than ping-pong mechanism; it accepts dCTP as well as CTP, making dCDP-diacylglycerol alongside CDP-diacylglycerol, and prefers phosphatidic acids carrying at least one unsaturated acyl chain. The reaction is reversible in vitro, but the physiological direction is activation of phosphatidic acid. The gene is essential and cannot be deleted; conditional mutants stop making phosphatidylethanolamine and phosphatidylglycerol and accumulate phosphatidic acid to about a third of the membrane lipid. Beyond bulk phospholipid, CdsA also supplies the diacylglycerol-pyrophosphate unit of MPIase, the glycolipid that drives membrane protein integration at the inner membrane, and its paralogue YnbB backs up that branch specifically. CdsA belongs to the prokaryotic-type CDS family (Pfam PF01148), which is distinct in lineage though not in chemistry from the mitochondrial Tam41-type CDP-diacylglycerol synthases. existing_annotations: - term: id: GO:0004605 label: phosphatidate cytidylyltransferase activity evidence_type: EXP original_reference_id: PMID:2995359 qualifier: enables review: summary: The defining activity, established on enzyme purified to near-homogeneity from an overproducing strain, with mechanism and kinetic constants. action: ACCEPT reason: This is the strongest row in the set and the basis of the EC assignment. Sparrow and Raetz purified the protein to about 90 percent homogeneity, matched its amino-acid composition to the product of the cloned cds gene, and characterised the reaction as sequential rather than ping-pong with surface-dilution kinetics in detergent, which is what an enzyme acting on a membrane-embedded lipid substrate should show. UniProt carries the catalytic activity with the same evidence code and PubMed identifier and gives the physiological direction as phosphatidic acid to CDP-diacylglycerol, so the measured reversibility in vitro is not a second function. Core. supported_by: - reference_id: PMID:2995359 supporting_text: has been purified to 90% homogeneity from Escherichia coli cells reference_section_type: ABSTRACT - reference_id: PMID:2995359 supporting_text: the enzyme mechanism is sequential, and computer-calculated kinetic constants are reported herein reference_section_type: ABSTRACT - reference_id: file:ECOLI/cdsA/cdsA-uniprot.txt record_section: ["CC:CATALYTIC ACTIVITY"] supporting_text: PhysiologicalDirection=left-to-right - term: id: GO:0004605 label: phosphatidate cytidylyltransferase activity evidence_type: IDA original_reference_id: PMID:30751 qualifier: enables review: summary: The same activity, assayed on enzyme partially purified from the cell envelope seven years earlier; it also establishes dCTP use and the reverse pyrophosphorolysis. action: ACCEPT reason: Langley and Kennedy solubilised the activity from the cell envelope with digitonin and purified it about 70-fold, reaching the same sequential mechanism and showing by isotopic exchange that pyrophosphate leaves before CDP-diglyceride. They also showed that one enzyme makes both CDP- and dCDP-diglyceride, CTP and dCTP being competitive substrates, which is why the single GO term covers both products. The row reinforces the EXP row rather than duplicating it, and the two are consistent. Core. supported_by: - reference_id: PMID:30751 supporting_text: was solubilized from the cell envelope by a differential extraction procedure involving the detergent digitonin reference_section_type: ABSTRACT - reference_id: PMID:30751 supporting_text: It therefore appears that a single enzyme catalyzes synthesis of both CDP-diglyceride and dCDP-diglyceride in E. coli. reference_section_type: ABSTRACT - term: id: GO:0004605 label: phosphatidate cytidylyltransferase activity evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: enables supporting_entities: - PANTHER:PTN000349517 - UniProtKB:P0ABG1 review: summary: Phylogenetic inference at a node of PTHR46382 whose descendant evidence includes this gene's own purified-enzyme annotations; the inherited activity is the right one. action: ACCEPT reason: What is tested here is the node placement, not the length of the WITH/FROM list. P0ABG1 is a member of PTHR46382 subfamily SF1 by its own DR PANTHER line and by the cached family entry list, and the activity asserted at the node is exactly the reaction the E. coli protein was purified and assayed for, so the gene lies inside the clade that carries the function and there is no sign of divergence or loss. The gene appearing in its own WITH/FROM is expected and is a marker that the node rests on experimental grounding in this very organism, not circularity, so nothing here is marked redundant. Note that this node, PTN000349517, is distinct from PTN000349519, the node used for the eukaryotic CDS enzymes in the same family (seen in the S. cerevisiae CDS1 annotations), so this is not a eukaryotic transfer. The IBD itself could not be read locally, since interpro/panther/PTHR46382 holds no -paint.tsv, so no claim is made about where in the tree the curator placed it. Core. supported_by: - reference_id: file:ECOLI/cdsA/cdsA-uniprot.txt record_section: ["DR:PROSITE"] supporting_text: 'DR PROSITE; PS01315; CDS; 1.' - reference_id: file:ECOLI/cdsA/cdsA-uniprot.txt record_section: ["CC:SIMILARITY"] supporting_text: Belongs to the CDS family. - reference_id: file:interpro/panther/PTHR46382/PTHR46382-entries.csv supporting_text: P0ABG1,Phosphatidate cytidylyltransferase,protein,83333,Escherichia coli (strain K12),Escherichia coli (strain K12),cdsA,285,PTHR46382:SF1,PHOSPHATIDATE CYTIDYLYLTRANSFERASE,True propagation_review: root_cause: NO_FAILURE_CORE source_entities: - source_id: PANTHER:PTN000349517 source_status: SUPPORTS_TRANSFER comment: A node of PTHR46382, the phosphatidate cytidylyltransferase family, asserting the family's defining reaction. The target is a confirmed member of the family and of its only subfamily, SF1, and has its own purified-enzyme evidence for the reaction, so inheritance and direct evidence agree. The node is not the eukaryotic CDS node PTN000349519. - source_id: UniProtKB:P0ABG1 source_status: SUPPORTS_TRANSFER comment: The target's own experimental annotation, appearing among the descendant evidences the PAINT curator used. Expected and not circular. - term: id: GO:0004605 label: phosphatidate cytidylyltransferase activity evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: enables supporting_entities: - RHEA:16229 - EC:2.7.7.41 review: summary: Automatic mapping from the Rhea reaction and EC number; both describe the assayed reaction exactly. action: ACCEPT reason: RHEA:16229 is the reaction UniProt records for this entry with experimental evidence from the purification paper, and EC 2.7.7.41 is carried on the entry with the same evidence code and PubMed identifier rather than as a bare name. The electronic row therefore restates a claim that is independently established for this protein, not a prediction, and it is consistent with the EXP, IDA and IBA rows for the same term. Core. supported_by: - reference_id: PMID:2995359 supporting_text: has been purified to 90% homogeneity from Escherichia coli cells reference_section_type: ABSTRACT - reference_id: file:ECOLI/cdsA/cdsA-uniprot.txt record_section: ["CC:PATHWAY"] supporting_text: 'diacylglycerol from sn-glycerol 3-phosphate: step 3/3.' - term: id: GO:0016772 label: transferase activity, transferring phosphorus-containing groups evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: enables supporting_entities: - InterPro:IPR000374 review: summary: A true but uninformative grandparent of the specific activity; refine to the cytidylyltransferase term the gene already carries. action: MODIFY reason: IPR000374 is the phosphatidate cytidylyltransferase signature, so the InterPro2GO mapping is right about the chemistry, but it stops at a very general class that says only that the protein moves a phosphorus-containing group. The specific term GO:0004605 is an is_a descendant of this one, is asserted on four other rows of this review, and is supported for this protein by purified-enzyme assays, so this is a refinement to a term the gene already carries rather than a correction. Not REMOVE, because the claim is true and the mapping is sound. proposed_replacement_terms: - id: GO:0004605 label: phosphatidate cytidylyltransferase activity supported_by: - reference_id: file:ECOLI/cdsA/cdsA-uniprot.txt record_section: ["DR:Pfam"] supporting_text: 'DR Pfam; PF01148; CTP_transf_1; 1.' - reference_id: PMID:2995359 supporting_text: has been purified to 90% homogeneity from Escherichia coli cells reference_section_type: ABSTRACT - term: id: GO:0016024 label: CDP-diacylglycerol biosynthetic process evidence_type: IDA original_reference_id: PMID:30751 qualifier: acts_upstream_of_or_within review: summary: The process the enzyme's own reaction constitutes; correct term, but the qualifier understates what was shown. action: ACCEPT reason: The assay in this paper is the synthesis of CDP-diglyceride itself from phosphatidic acid and CTP by enzyme solubilised from the cell envelope, so CdsA does not merely act upstream of the process, it performs it. The term is right and is the gene's core process. The weaker qualifier is recorded as a source defect against EcoCyc rather than used as a reason to change or drop the term, and the action is kept consistent with the three other rows for this term. supported_by: - reference_id: PMID:30751 supporting_text: The cytosine liponucleotides CDP-diglyceride and dCDP-diglyceride are key intermediates in phospholipid biosynthesis in Escherichia coli reference_section_type: ABSTRACT - reference_id: PMID:30751 supporting_text: was solubilized from the cell envelope by a differential extraction procedure involving the detergent digitonin reference_section_type: ABSTRACT source_defects: - defect_type: WRONG_QUALIFIER resource: ECOCYC slot: Qualifier requested_change: REPOINT asserted_value: acts_upstream_of_or_within corrected_value: involved_in confidence: PROBABLE explanation: This IDA row rests on a direct enzyme assay in which the gene product catalyses the formation of CDP-diglyceride from phosphatidic acid and CTP. A gene product that catalyses the committed reaction of a biosynthetic process is involved_in that process; acts_upstream_of_or_within is for cases where direct participation is not established, and it is the weaker of the two available qualifiers. The same database's IMP row for this term uses the same qualifier, which is more defensible there because that evidence is a mutant phenotype. Confidence is PROBABLE rather than CONFIRMED because the cached publication entry is abstract-only, so the full text the curator worked from could not be read. supported_by: - reference_id: PMID:30751 supporting_text: The enzyme utilized both CTP and dCTP as nucleotide substrate reference_section_type: ABSTRACT - term: id: GO:0016024 label: CDP-diacylglycerol biosynthetic process evidence_type: IMP original_reference_id: PMID:6273438 qualifier: acts_upstream_of_or_within review: summary: Conditional cds mutants accumulate phosphatidic acid and deplete the liponucleotide pool, which is the in vivo demonstration that this gene makes CDP-diacylglycerol. action: ACCEPT reason: Ganong and Raetz isolated conditionally lethal cds mutants in which phosphatidic acid rises to about 30 percent of membrane lipid above pH 8 while phosphatidylethanolamine and phosphatidylglycerol synthesis stops more than ten-fold, and in which the liponucleotide pool falls to about a seventh of wild type. Reversion restores both growth and enzyme activity in 21 of 26 revertants, which ties the phenotype to the gene rather than to a linked lesion. This is the in vivo counterpart of the enzymology and the reason the process term is core rather than merely plausible. supported_by: - reference_id: PMID:6273438 supporting_text: Above pH 8, GN80 accumulates phosphatidic acid to about 30% of the total membrane lipid, while the de novo syntheses of phosphatidylethanolamine and phosphatidylglycerol are abruptly inhibited by over 10-fold. reference_section_type: ABSTRACT - reference_id: PMID:6273438 supporting_text: the liponucleotide pool of GN80 is about one-seventh that of an isogenic wild type reference_section_type: ABSTRACT - term: id: GO:0016024 label: CDP-diacylglycerol biosynthetic process evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: involved_in supporting_entities: - PANTHER:PTN000349517 - UniProtKB:P0ABG1 review: summary: The process that follows from the inherited activity, asserted at the same PTHR46382 node; consistent with the experimental rows. action: ACCEPT reason: This is the process counterpart of the molecular-function IBA at PTN000349517 and stands or falls with it. Since the target is a member of the family and has direct evidence for both the reaction and the in vivo consequence of losing it, the inherited process claim is sound, and the qualifier here, involved_in, is the one the EcoCyc rows should also carry. The target in its own WITH/FROM is expected. Core. supported_by: - reference_id: PMID:6273438 supporting_text: Our results constitute definitive physiological proof that CDP-diglyceride is an obligatory precursor for over 90% of the phosphatidylethanolamine and phosphatidylglycerol in E. coli. reference_section_type: ABSTRACT - reference_id: file:ECOLI/cdsA/cdsA-uniprot.txt record_section: ["CC:SIMILARITY"] supporting_text: Belongs to the CDS family. propagation_review: root_cause: NO_FAILURE_CORE source_entities: - source_id: PANTHER:PTN000349517 source_status: SUPPORTS_TRANSFER comment: Same node as the molecular-function row. The process asserted is the one the family's reaction constitutes, and the target has its own mutant-phenotype evidence for it, so the inference is corroborated rather than merely inherited. - source_id: UniProtKB:P0ABG1 source_status: SUPPORTS_TRANSFER comment: The target's own experimental grounding among the descendant evidences; expected, not circular. - term: id: GO:0016024 label: CDP-diacylglycerol biosynthetic process evidence_type: IEA original_reference_id: GO_REF:0000041 qualifier: involved_in supporting_entities: - UniPathway:UPA00557 review: summary: Pathway-vocabulary mapping that places the enzyme at the last step of CDP-diacylglycerol biosynthesis; correct and consistent with the experimental rows. action: ACCEPT reason: UPA00557 is CDP-diacylglycerol biosynthesis, and the UniProt entry assigns this protein step 3 of 3 of that pathway from sn-glycerol 3-phosphate, with the assignment traced to the purification paper. The electronic row therefore agrees with the IDA, IMP and IBA rows for the same term and adds the pathway position. Core. supported_by: - reference_id: file:ECOLI/cdsA/cdsA-uniprot.txt record_section: ["CC:PATHWAY"] supporting_text: 'diacylglycerol from sn-glycerol 3-phosphate: step 3/3.' - reference_id: PMID:6273438 supporting_text: Our results constitute definitive physiological proof that CDP-diglyceride is an obligatory precursor for over 90% of the phosphatidylethanolamine and phosphatidylglycerol in E. coli. reference_section_type: ABSTRACT - term: id: GO:0005886 label: plasma membrane evidence_type: IDA original_reference_id: PMID:15919996 qualifier: located_in review: summary: Direct inner-membrane assignment from the genome-wide topology study that also fixed this protein's C-terminus in the periplasm. action: ACCEPT reason: Daley and colleagues tagged inner membrane proteins with alkaline phosphatase and GFP and determined the side of the membrane on which each C-terminus lies, then built topology models constrained by that data. UniProt cites this work with an experimental evidence code both for the subcellular location of this entry and for its single experimentally supported topological domain, the periplasmic C-terminal residue. In a diderm bacterium the inner membrane is the plasma membrane, so GO:0005886 is the correct term and no more specific bacterial membrane term is needed. Core, since the enzyme's substrate and product are membrane lipids. supported_by: - reference_id: PMID:15919996 supporting_text: we established the periplasmic or cytoplasmic locations of the C termini for 601 inner membrane proteins reference_section_type: ABSTRACT - reference_id: file:ECOLI/cdsA/cdsA-uniprot.txt record_section: ["CC:SUBCELLULAR LOCATION"] supporting_text: 'SUBCELLULAR LOCATION: Cell inner membrane' - term: id: GO:0005886 label: plasma membrane evidence_type: IDA original_reference_id: PMID:30751 qualifier: located_in review: summary: The activity was extracted from the cell envelope and required detergent throughout, which places the enzyme in the membrane independently of the topology study. action: ACCEPT reason: This 1978 paper obtained the cytidylyltransferase by differential digitonin extraction of the cell envelope and purified it only in the presence of detergent. That is a membrane location established biochemically, twenty-seven years before the topology survey, and the two agree. The cached entry is abstract-only, so no claim is made here about which envelope sub-fraction was resolved; the inner-membrane assignment rests on the other IDA row and on UniProt. Consistent with the other plasma membrane rows. supported_by: - reference_id: PMID:30751 supporting_text: was solubilized from the cell envelope by a differential extraction procedure involving the detergent digitonin reference_section_type: ABSTRACT - reference_id: PMID:2995359 supporting_text: The purification required the use of different detergents at each step, illustrating the refractory hydrophobic nature of this protein. reference_section_type: ABSTRACT - term: id: GO:0005886 label: plasma membrane evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: is_active_in supporting_entities: - PANTHER:PTN000349517 - UniProtKB:P0ABG1 review: summary: Phylogenetic assertion that the family's activity takes place in the plasma membrane; the is_active_in qualifier is the right one for this enzyme. action: ACCEPT reason: The substrate, phosphatidic acid, and the product, CDP-diacylglycerol, are both membrane lipids, and the purified enzyme shows surface-dilution kinetics, meaning its rate depends on substrate surface concentration in the bilayer or micelle rather than on bulk concentration. The catalysis therefore happens at the membrane and is_active_in is stronger and more informative than located_in here. The node assertion is inherited within the family rather than transferred from eukaryotes, which locate the same activity in the endoplasmic reticulum under a different node. Core. supported_by: - reference_id: PMID:2995359 supporting_text: The pure enzyme displays surface dilution kinetics when assayed in the presence of Triton X-100. reference_section_type: ABSTRACT - reference_id: file:ECOLI/cdsA/cdsA-uniprot.txt record_section: ["CC:SUBCELLULAR LOCATION"] supporting_text: 'SUBCELLULAR LOCATION: Cell inner membrane' propagation_review: root_cause: NO_FAILURE_CORE source_entities: - source_id: PANTHER:PTN000349517 source_status: SUPPORTS_TRANSFER comment: The node is in the prokaryotic part of PTHR46382, not the eukaryotic CDS node PTN000349519 that carries endoplasmic reticulum membrane, so the compartment asserted matches the target's own cell plan and its experimentally determined location. - source_id: UniProtKB:P0ABG1 source_status: SUPPORTS_TRANSFER comment: The target's own direct localisation evidence among the descendant evidences; expected, not circular. - term: id: GO:0005886 label: plasma membrane evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in supporting_entities: - UniProtKB-SubCell:SL-0037 review: summary: Mapping of the curated UniProt subcellular location, cell inner membrane, onto the nearest GO term; correct for a Gram-negative bacterium. action: ACCEPT reason: The UniProt location statement this row maps from carries an experimental evidence code citing the topology study, so this is not an unvetted prediction being recycled but a curated experimental location passed through a controlled-vocabulary mapping. Cell inner membrane maps conservatively to plasma membrane, which is the correct GO term for the E. coli cytoplasmic membrane, and the row agrees with the two IDA rows. Core. supported_by: - reference_id: file:ECOLI/cdsA/cdsA-uniprot.txt record_section: ["CC:SUBCELLULAR LOCATION"] supporting_text: 'SUBCELLULAR LOCATION: Cell inner membrane' - reference_id: PMID:15919996 supporting_text: we report a global topology analysis of the Escherichia coli inner membrane proteome reference_section_type: ABSTRACT - term: id: GO:0016020 label: membrane evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: located_in supporting_entities: - InterPro:IPR000374 review: summary: True but less specific than the evidence supports; refine to the plasma membrane term the gene already carries on four other rows. action: MODIFY reason: A generic membrane term is not a root organelle term, so the organelle policy does not protect it, and the rule that a correct but general term should be refined applies. The compartment is known experimentally for this protein, the inner membrane of E. coli, which is its plasma membrane, and GO:0005886 is a descendant of GO:0016020 already asserted here by two IDA rows, an IBA and a UniProt mapping. The InterPro signature itself is the right family signature, so this is a refinement and not a correction, and REMOVE would be wrong. proposed_replacement_terms: - id: GO:0005886 label: plasma membrane supported_by: - reference_id: file:ECOLI/cdsA/cdsA-uniprot.txt record_section: ["CC:SUBCELLULAR LOCATION"] supporting_text: 'SUBCELLULAR LOCATION: Cell inner membrane' - reference_id: PMID:15919996 supporting_text: we established the periplasmic or cytoplasmic locations of the C termini for 601 inner membrane proteins reference_section_type: ABSTRACT - term: id: GO:0009247 label: glycolipid biosynthetic process evidence_type: IMP original_reference_id: PMID:30718729 qualifier: involved_in review: summary: Proposed addition. Beyond bulk phospholipid, CdsA is required for and contributes the lipid unit of MPIase, the glycolipid that drives membrane protein integration. action: NEW reason: The entity performing the step is CdsA itself, which is what distinguishes this from a mere requirement. Depleting CdsA leaves MPIase undetectable, overproducing it raises MPIase about three-fold, and inverted vesicles from CdsA overproducers, as well as partially purified CdsA8, convert radiolabelled phosphatidic acid plus CTP plus N-acetylglucosamine-1-phosphate into the first MPIase intermediate, identified against a synthetic reference by thin-layer chromatography in two solvent systems and by mass spectrometry. The paralogue YnbB restores MPIase but not phospholipid synthesis, and both genes are described as rate-limiting for MPIase, so the branch is real and not an artefact of CDP-diacylglycerol shortage alone. UniProt states the role in its FUNCTION line with an experimental evidence code. MPIase has no GO term of its own and the general glycolipid biosynthesis term is the closest fit. This is non-core, because the gene's defining activity is phosphatidate cytidylyltransferase, and this branch consumes a small fraction of its output. Deliberately not proposed is any protein integration or translocation process term, since MPIase, not CdsA, acts on the substrate protein. supported_by: - reference_id: PMID:30718729 supporting_text: We found that MPIase was hardly detected in both the cdsA and cdsA/ynbB knockouts reference_section_type: RESULTS - reference_id: PMID:30718729 supporting_text: These observations indicate that CdsA is a biosynthetic enzyme for MPIase. reference_section_type: RESULTS - reference_id: PMID:30739787 supporting_text: Under CdsA-depleted conditions, YnbB overproduction restored MPIase expression, but not phospholipid biosynthesis. reference_section_type: ABSTRACT - reference_id: file:ECOLI/cdsA/cdsA-uniprot.txt record_section: ["CC:FUNCTION"] supporting_text: of MPIase, a glycolipid essential for membrane protein integration and core_functions: - description: Activates phosphatidic acid with CTP to form CDP-diacylglycerol and pyrophosphate at the inner membrane, the single committed step between phosphatidic acid and the liponucleotide from which phosphatidylglycerol, cardiolipin and phosphatidylethanolamine are all built. The enzyme is polytopic and acts on membrane-embedded substrate with surface-dilution kinetics by a sequential mechanism, and it accepts dCTP as well as CTP, so it is also the source of dCDP-diacylglycerol. It is essential, and when it is limiting phosphatidic acid accumulates while head-group phospholipid synthesis stops. molecular_function: id: GO:0004605 label: phosphatidate cytidylyltransferase activity directly_involved_in: - id: GO:0016024 label: CDP-diacylglycerol biosynthetic process locations: - id: GO:0005886 label: plasma membrane substrates: - id: CHEBI:58608 label: 1,2-diacyl-sn-glycerol 3-phosphate(2-) - id: CHEBI:37563 label: CTP(4-) supported_by: - reference_id: PMID:2995359 supporting_text: The pure enzyme displays surface dilution kinetics when assayed in the presence of Triton X-100. reference_section_type: ABSTRACT - reference_id: PMID:6273438 supporting_text: Our results constitute definitive physiological proof that CDP-diglyceride is an obligatory precursor for over 90% of the phosphatidylethanolamine and phosphatidylglycerol in E. coli. reference_section_type: ABSTRACT - reference_id: file:ECOLI/cdsA/cdsA-uniprot.txt record_section: ["CC:PATHWAY"] supporting_text: 'diacylglycerol from sn-glycerol 3-phosphate: step 3/3.' references: - id: GO_REF:0000002 title: Gene Ontology annotation through association of InterPro records with GO terms findings: [] - id: GO_REF:0000033 title: Annotation inferences using phylogenetic trees findings: [] - id: GO_REF:0000041 title: Gene Ontology annotation based on UniPathway vocabulary mapping findings: [] - id: GO_REF:0000044 title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt findings: [] - id: GO_REF:0000120 title: Combined Automated Annotation using Multiple IEA Methods findings: [] - id: PMID:2995359 title: Purification and properties of the membrane-bound CDP-diglyceride synthetase from Escherichia coli. findings: - statement: The enzyme was purified to about 90 percent homogeneity from an overproducing strain, its amino-acid composition matching the product of the cloned cds gene, and it required a different detergent at each purification step. supporting_text: has been purified to 90% homogeneity from Escherichia coli cells reference_section_type: ABSTRACT - statement: Purified enzyme shows surface-dilution kinetics in Triton X-100 and a sequential rather than ping-pong mechanism, with kinetic constants reported. supporting_text: The pure enzyme displays surface dilution kinetics when assayed in the presence of Triton X-100. reference_section_type: ABSTRACT - statement: The hydrophobic, detergent-dependent behaviour of the protein through every purification step is consistent with an integral membrane protein. supporting_text: The purification required the use of different detergents at each step, illustrating the refractory hydrophobic nature of this protein. reference_section_type: ABSTRACT reference_review: relevance: HIGH correctness: VERIFIED review_notes: PubMed-verified; this is the paper UniProt cites with ECO:0000269 for FUNCTION, for the catalytic activity and for the EC number, and the paper the GOA EXP row rests on. The cached entry is abstract-only, so only abstract-level statements are quoted and no claim here depends on a figure. - id: PMID:30751 full_text_unavailable: true title: Partial purification and properties of CTP:phosphatidic acid cytidylyltransferase from membranes of Escherichia coli. findings: - statement: The activity was solubilised from the cell envelope with digitonin and purified about 70-fold, establishing a membrane location biochemically. supporting_text: was solubilized from the cell envelope by a differential extraction procedure involving the detergent digitonin reference_section_type: ABSTRACT - statement: One enzyme makes both CDP-diglyceride and dCDP-diglyceride, CTP and dCTP being competitive substrates, so the single GO term covers both products. supporting_text: It therefore appears that a single enzyme catalyzes synthesis of both CDP-diglyceride and dCDP-diglyceride in E. coli. reference_section_type: ABSTRACT - statement: The enzyme also catalyses the reverse pyrophosphorolysis in vitro; UniProt records the physiological direction as phosphatidic acid to CDP-diacylglycerol, so this is not a second function. supporting_text: The enzyme also catalyzes a pyrophosphorolysis of CDP-diglyceride reference_section_type: ABSTRACT reference_review: relevance: HIGH correctness: VERIFIED review_notes: PubMed-verified; EcoCyc uses it for three rows (activity, plasma membrane, CDP-diacylglycerol biosynthesis), which is consistent with what the abstract reports. Full text could not be retrieved in this session, PMC218635 returning 403, so the review works from the abstract and does not second-guess which envelope sub-fraction the curator saw. - id: PMID:6273438 title: Massive accumulation of phosphatidic acid in conditionally lethal CDP-diglyceride synthetase mutants and cytidine auxotrophs of Escherichia coli. findings: - statement: Conditionally lethal cds mutants accumulate phosphatidic acid to about 30 percent of membrane lipid above pH 8 while phosphatidylethanolamine and phosphatidylglycerol synthesis is inhibited more than ten-fold, and the liponucleotide pool falls to about a seventh of wild type. supporting_text: Above pH 8, GN80 accumulates phosphatidic acid to about 30% of the total membrane lipid, while the de novo syntheses of phosphatidylethanolamine and phosphatidylglycerol are abruptly inhibited by over 10-fold. reference_section_type: ABSTRACT - statement: The authors conclude that CDP-diglyceride is an obligatory precursor of more than 90 percent of the two major E. coli phospholipids, which is the in vivo basis for treating this reaction as the committed step. supporting_text: Our results constitute definitive physiological proof that CDP-diglyceride is an obligatory precursor for over 90% of the phosphatidylethanolamine and phosphatidylglycerol in E. coli. reference_section_type: ABSTRACT reference_review: relevance: HIGH correctness: VERIFIED review_notes: PubMed-verified and correctly cited by EcoCyc for the IMP row. Abstract-only in the cache. The genetic attribution is strengthened in the abstract itself by the report that 21 of 26 spontaneous revertants regain parental enzyme levels. - id: PMID:15919996 title: Global topology analysis of the Escherichia coli inner membrane proteome. findings: - statement: C-terminal alkaline phosphatase and GFP tagging fixed the periplasmic or cytoplasmic location of the C-terminus for 601 inner membrane proteins, and those constraints were used to derive topology models. supporting_text: we established the periplasmic or cytoplasmic locations of the C termini for 601 inner membrane proteins reference_section_type: ABSTRACT reference_review: relevance: HIGH correctness: VERIFIED review_notes: PubMed-verified. UniProt cites it with ECO:0000269 both for the subcellular location of P0ABG1 and for its single experimentally supported topological domain, the periplasmic C-terminal residue, which is what identifies b0175 as one of the 601 proteins. The abstract reports the method and scale only, so the per-protein result is taken from the UniProt entry rather than read directly. - id: PMID:30718729 title: CdsA is involved in biosynthesis of glycolipid MPIase essential for membrane protein integration in vivo. findings: - statement: Depleting CdsA leaves MPIase undetectable and blocks M13 procoat processing, while overproducing CdsA raises MPIase about three-fold. supporting_text: We found that MPIase was hardly detected in both the cdsA and cdsA/ynbB knockouts reference_section_type: RESULTS - statement: Inverted vesicles from CdsA overproducers and partially purified CdsA8 convert labelled phosphatidic acid, CTP and GlcNAc-1-phosphate into the first MPIase intermediate, identified against a synthetic reference by chromatography and mass spectrometry, with CDP-diacylglycerol apparently not released from the enzyme. supporting_text: These observations strongly suggest that CDP-DAG is synthesized and then converted to compound I on CdsA. reference_section_type: RESULTS - statement: cdsA is essential; neither the knockout nor the cdsA ynbB double knockout grows without induction of the complementing plasmid. supporting_text: both the cdsA knockout and the cdsA/ynbB double knockout were unable to grow in the absence of inducer arabinose reference_section_type: RESULTS reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text retrieved from PMC and read; UniProt cites it with ECO:0000269 for FUNCTION and DISRUPTION PHENOTYPE. The compound I chemistry is shown with inverted vesicles and with the CdsA8 (Y207H) mutant at non-permissive pH rather than with wild-type purified enzyme, which is why this review asserts a process term and not a molecular function for that step. Single-laboratory result, not independently replicated. - id: PMID:30739787 title: YnbB is a CdsA paralogue dedicated to biosynthesis of glycolipid MPIase involved in membrane protein integration. findings: - statement: Under CdsA depletion, YnbB overproduction restores MPIase but not phospholipid synthesis, and chimera analysis attributes the fast CDP-diacylglycerol synthesis needed for growth to the N-terminal half of CdsA. supporting_text: Under CdsA-depleted conditions, YnbB overproduction restored MPIase expression, but not phospholipid biosynthesis. reference_section_type: ABSTRACT - statement: The authors read YnbB as a backup dedicated to the MPIase branch, which leaves the division of labour between the two paralogues in the wild type unresolved. supporting_text: the role of YnbB seems to be as a backup for MPIase biosynthesis reference_section_type: ABSTRACT reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: PubMed-verified and cited by UniProt with ECO:0000269 for FUNCTION and DOMAIN. Abstract-only in the cache. Same laboratory as PMID:30718729, so it corroborates the branch but is not independent of it. - id: PMID:30936205 title: Increased expression of the bacterial glycolipid MPIase is required for efficient protein translocation across membranes in cold conditions. findings: - statement: MPIase rises in the cold and the rise depends on cdsA or ynbB, which the authors describe as rate-limiting enzymes for MPIase biosynthesis. supporting_text: either the cdsA or ynbB gene, both encoding rate-limiting enzymes for MPIase biosynthesis, is responsible for the increase in the MPIase expression reference_section_type: ABSTRACT - statement: MPIase is the glycolipid that drives membrane protein integration into the E. coli cytoplasmic membrane, which is the entity that acts on the substrate protein, not CdsA. supporting_text: Membrane protein integrase (MPIase) is a glycolipid that drives membrane protein integration into the cytoplasmic membrane in Escherichia coli reference_section_type: ABSTRACT reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: PubMed-verified and cited by UniProt with ECO:0000269 for FUNCTION and INDUCTION. Abstract-only in the cache. Used here for the regulatory link and for the point that the integration activity belongs to MPIase; it is not used to support a protein-translocation annotation on CdsA. - id: PMID:2995358 title: Molecular cloning and sequencing of the gene for CDP-diglyceride synthetase of Escherichia coli. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: PubMed-verified companion paper to PMID:2995359, cited by UniProt for the gene name and the original sequence. Abstract-only in the cache; it establishes gene identity rather than function, so nothing in this review is quoted from it. - id: file:ECOLI/cdsA/cdsA-uniprot.txt title: UniProtKB entry P0ABG1 (CDSA_ECOLI) findings: - statement: Curated catalytic activity with Rhea 16229 and the physiological direction, pathway position, inner membrane location, essentiality and MPIase involvement, each tied to the primary literature by ECO:0000269. record_section: ["CC:CATALYTIC ACTIVITY"] supporting_text: PhysiologicalDirection=left-to-right - statement: The entry places the enzyme at the final step of CDP-diacylglycerol biosynthesis from sn-glycerol 3-phosphate. record_section: ["CC:PATHWAY"] supporting_text: 'diacylglycerol from sn-glycerol 3-phosphate: step 3/3.' - statement: The gene cannot be deleted, and depletion causes a phospholipid-synthesis defect together with MPIase loss and accumulation of membrane and secretory protein precursors. record_section: ["CC:DISRUPTION PHENOTYPE"] supporting_text: Essential, cannot be deleted (PubMed:30718729). - statement: Family placement is supported by independent signature resources, the CDS family note, the NCBIfam model for cdsA, Pfam CTP_transf_1 and PROSITE PS01315. record_section: ["DR:Pfam"] supporting_text: 'DR Pfam; PF01148; CTP_transf_1; 1.' reference_review: relevance: HIGH correctness: VERIFIED review_notes: Entry version 122 of 04-NOV-2026, as cached. Only curated statements with experimental evidence codes, family and signature cross-references, and sequence-analysis features were used as support; protein names, the EC number as a label, keywords and the record's own DR GO lines were not. - id: file:interpro/panther/PTHR46382/PTHR46382-entries.csv title: Cached PANTHER family entry list for PTHR46382 (phosphatidate cytidylyltransferase) findings: - statement: P0ABG1 is listed as a member of PTHR46382 and of its single subfamily SF1, alongside bacterial orthologues from B. subtilis, Aquifex and Chlamydia, which confirms the family placement the IBA rows depend on without relying on the GOA row itself. supporting_text: P0ABG1,Phosphatidate cytidylyltransferase,protein,83333,Escherichia coli (strain K12),Escherichia coli (strain K12),cdsA,285,PTHR46382:SF1,PHOSPHATIDATE CYTIDYLYLTRANSFERASE,True reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Read directly from the committed file. Note that this directory holds no -paint.tsv, so the IBD assertion at PTN000349517 could not be read locally and no claim is made about where in the tree it sits; the node identifier is taken only from the GOA WITH/FROM column. knowledge_gaps: - gap_statement: It is not known whether wild-type CdsA itself catalyses the transfer of N-acetylglucosamine 1-phosphate onto the diacylglycerol-pyrophosphate unit to make the first MPIase intermediate, or whether it only supplies CDP-diacylglycerol to a separate transferase, with the paralogue YnbB carrying the dedicated activity. boundary: What is established is that depleting CdsA abolishes detectable MPIase, that overproducing it raises MPIase, and that inverted vesicles from overproducers and partially purified CdsA8 form the intermediate from phosphatidic acid, CTP and N-acetylglucosamine 1-phosphate, with CDP-diacylglycerol apparently not released. What is not established is the same chemistry on purified wild-type enzyme under physiological conditions, since the clean in vitro demonstration used the Y207H CdsA8 mutant at a non-permissive high pH, and the separate finding that YnbB restores MPIase but not phospholipid synthesis leaves open which paralogue performs the step in a normally growing cell. gap_kind: - BIOLOGY dark_aspect: RESIDUAL_SUBGAP status: OPEN significance: This decides whether CdsA has one molecular function or two. If the transfer happens on CdsA, a GO molecular function term for the activity is needed, because no existing term describes it, and the MPIase branch would be a second catalytic activity of an enzyme otherwise treated as doing one thing. If it does not, the correct picture is that CdsA supplies the precursor and the process annotation should eventually be narrowed or moved. The review asserts the process term and deliberately proposes no molecular function term until this is settled. resolution: Reconstitute purified wild-type CdsA, purified YnbB and the two chimeras in proteoliposomes and measure formation of the intermediate from defined substrates at physiological pH, with and without added N-acetylglucosamine 1-phosphate, testing directly whether CDP-diacylglycerol is released as a free intermediate. provenance: - reference_id: PMID:30718729 supporting_text: These observations strongly suggest that CDP-DAG is synthesized and then converted to compound I on CdsA. - reference_id: PMID:30718729 supporting_text: Sequencing of the cdsA8 allele revealed that this mutation comprises an amino acid substitution of Tyr 207 to His - reference_id: PMID:30739787 supporting_text: the role of YnbB seems to be as a backup for MPIase biosynthesis - gap_statement: The catalytic site of CdsA has not been localised to a face of the inner membrane, and no catalytic residues or metal requirement have been identified for the E. coli enzyme. boundary: What is established is that the protein is polytopic, with eight predicted helices and an experimentally determined periplasmic C-terminus, and that the reaction is sequential with surface-dilution kinetics, so catalysis occurs at the lipid interface. What is not established is which residues form the active site, on which side of the bilayer the nucleotide binds, or whether a divalent metal is required; the UniProt entry carries no binding, metal or active-site feature, and the only metal-related observation is an unexplained physical effect of EDTA during purification. gap_kind: - BIOLOGY dark_aspect: RESIDUAL_SUBGAP status: OPEN significance: Both substrates are charged and one is cytoplasmic while the lipid is bilayer- embedded, so the geometry constrains how phosphatidic acid reaches the site and whether the enzyme is a candidate for the kind of side-specific component term GO can express. It also bears on whether a cofactor annotation is warranted; this review asserts none. resolution: Determine a structure of E. coli CdsA with CTP and a phosphatidic acid analogue, or map the site by cysteine-scanning accessibility and mutagenesis of candidate residues, and test the divalent metal requirement of purified enzyme with chelators and defined metal add-back. provenance: - reference_id: PMID:2995359 supporting_text: The pure enzyme displays surface dilution kinetics when assayed in the presence of Triton X-100. - reference_id: file:ECOLI/cdsA/cdsA-uniprot.txt supporting_text: Multi-pass membrane protein suggested_questions: - question: How should integral versus peripheral membrane topology now be expressed, given that GO:0005887 integral component of plasma membrane and GO:0016021 integral component of membrane have been merged away into GO:0005886 and GO:0016020? CdsA is polytopic in the inner membrane, with its C-terminus placed in the periplasm experimentally, eight predicted transmembrane helices and purification that needed a different detergent at every step, yet after the topology merge the ontology offers no term that distinguishes this from a peripherally associated plasma-membrane protein. experts: - GO cellular component curators - question: Does GO want a molecular function term for formation of the N-acetylglucosamine pyrophosphoryl-diacylglycerol unit of MPIase, and should such a term wait until it is clear whether CdsA, YnbB or a separate transferase performs the step in vivo? experts: - GO molecular function curators - bacterial membrane biogenesis researchers - question: Should the glycolipid MPIase have process terms of its own, given that it is a characterised E. coli glycolipid with a dedicated biosynthetic branch and a defined role in membrane protein integration, rather than being annotated only through the generic glycolipid biosynthesis term? experts: - GO biological process curators - protein translocation researchers - question: Should EcoCyc's two CDP-diacylglycerol biosynthesis rows for this gene be requalified from acts_upstream_of_or_within to involved_in, given that the gene product catalyses the reaction that constitutes the process? experts: - EcoCyc curators - GO Consortium biocurators suggested_experiments: - hypothesis: Wild-type CdsA forms the first MPIase intermediate on the enzyme without releasing CDP-diacylglycerol, rather than merely supplying CDP-diacylglycerol to a separate transferase. description: Purify wild-type CdsA, the CdsA8 mutant, YnbB and the two reciprocal chimeras, reconstitute each into proteoliposomes of defined composition, and follow formation of CDP-diacylglycerol and of the N-acetylglucosamine pyrophosphoryl-diacylglycerol intermediate from radiolabelled phosphatidic acid at physiological pH, including pulse-chase and trapping experiments that test whether free CDP-diacylglycerol is an obligatory released intermediate. experiment_type: reconstitution of purified enzymes in proteoliposomes with radiolabelled substrates - hypothesis: CdsA requires a divalent metal ion for catalysis, as the EDTA sensitivity observed during purification suggests. description: Assay purified CdsA after dialysis against chelator, then add back magnesium, manganese and calcium individually over a concentration range, measuring initial rates with defined phosphatidic acid and CTP concentrations in mixed micelles, and determine by inductively coupled plasma mass spectrometry whether the purified enzyme carries bound metal. experiment_type: enzyme kinetics with chelator treatment and metal add-back - hypothesis: The active site of CdsA faces the cytoplasm, as required if cytoplasmic CTP is a substrate. description: Use substituted-cysteine accessibility mapping on a cysteine-less CdsA background, probing from each side of the inner membrane in intact cells, spheroplasts and inverted vesicles, to assign the loops that carry the conserved family motif to one face, and test candidate catalytic residues by mutagenesis with complementation of a cdsA depletion strain. experiment_type: substituted-cysteine accessibility mapping with complementation assays