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.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004605 phosphatidate cytidylyltransferase activity | EXP PMID:2995359 Purification and properties of the membrane-bound CDP-diglyc... | ACCEPT | Summary: The defining activity, established on enzyme purified to near-homogeneity from an overproducing strain, with mechanism and kinetic constants. 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. Supporting Evidence: PMID:2995359 has been purified to 90% homogeneity from Escherichia coli cells PMID:2995359 the enzyme mechanism is sequential, and computer-calculated kinetic constants are reported herein file:ECOLI/cdsA/cdsA-uniprot.txt PhysiologicalDirection=left-to-right |
| GO:0004605 phosphatidate cytidylyltransferase activity | IDA PMID:30751 Partial purification and properties of CTP:phosphatidic acid... | ACCEPT | 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. 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. Supporting Evidence: PMID:30751 was solubilized from the cell envelope by a differential extraction procedure involving the detergent digitonin PMID:30751 It therefore appears that a single enzyme catalyzes synthesis of both CDP-diglyceride and dCDP-diglyceride in E. coli. |
| GO:0004605 phosphatidate cytidylyltransferase activity | IBA GO_REF:0000033 | ACCEPT | 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. 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. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000349517 SUPPORTS TRANSFER 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. UniProtKB:P0ABG1 SUPPORTS TRANSFER The target's own experimental annotation, appearing among the descendant evidences the PAINT curator used. Expected and not circular. Supporting Evidence: file:ECOLI/cdsA/cdsA-uniprot.txt DR PROSITE; PS01315; CDS; 1. file:ECOLI/cdsA/cdsA-uniprot.txt Belongs to the CDS family. file:interpro/panther/PTHR46382/PTHR46382-entries.csv P0ABG1,Phosphatidate cytidylyltransferase,protein,83333,Escherichia coli (strain K12),Escherichia coli (strain K12),cdsA,285,PTHR46382:SF1,PHOSPHATIDATE CYTIDYLYLTRANSFERASE,True |
| GO:0004605 phosphatidate cytidylyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Automatic mapping from the Rhea reaction and EC number; both describe the assayed reaction exactly. 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. Supporting Evidence: PMID:2995359 has been purified to 90% homogeneity from Escherichia coli cells file:ECOLI/cdsA/cdsA-uniprot.txt diacylglycerol from sn-glycerol 3-phosphate: step 3/3. |
| GO:0016772 transferase activity, transferring phosphorus-containing groups | IEA GO_REF:0000002 | MODIFY | Summary: A true but uninformative grandparent of the specific activity; refine to the cytidylyltransferase term the gene already carries. 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 replacements: phosphatidate cytidylyltransferase activity Supporting Evidence: file:ECOLI/cdsA/cdsA-uniprot.txt DR Pfam; PF01148; CTP_transf_1; 1. PMID:2995359 has been purified to 90% homogeneity from Escherichia coli cells |
| GO:0016024 CDP-diacylglycerol biosynthetic process | IDA PMID:30751 Partial purification and properties of CTP:phosphatidic acid... | ACCEPT | Summary: The process the enzyme's own reaction constitutes; correct term, but the qualifier understates what was shown. 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. Supporting Evidence: PMID:30751 The cytosine liponucleotides CDP-diglyceride and dCDP-diglyceride are key intermediates in phospholipid biosynthesis in Escherichia coli PMID:30751 was solubilized from the cell envelope by a differential extraction procedure involving the detergent digitonin |
| GO:0016024 CDP-diacylglycerol biosynthetic process | IMP PMID:6273438 Massive accumulation of phosphatidic acid in conditionally l... | ACCEPT | Summary: Conditional cds mutants accumulate phosphatidic acid and deplete the liponucleotide pool, which is the in vivo demonstration that this gene makes CDP-diacylglycerol. 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. Supporting Evidence: PMID:6273438 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. PMID:6273438 the liponucleotide pool of GN80 is about one-seventh that of an isogenic wild type |
| GO:0016024 CDP-diacylglycerol biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: The process that follows from the inherited activity, asserted at the same PTHR46382 node; consistent with the experimental rows. 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. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000349517 SUPPORTS TRANSFER 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. UniProtKB:P0ABG1 SUPPORTS TRANSFER The target's own experimental grounding among the descendant evidences; expected, not circular. Supporting Evidence: PMID:6273438 Our results constitute definitive physiological proof that CDP-diglyceride is an obligatory precursor for over 90% of the phosphatidylethanolamine and phosphatidylglycerol in E. coli. file:ECOLI/cdsA/cdsA-uniprot.txt Belongs to the CDS family. |
| GO:0016024 CDP-diacylglycerol biosynthetic process | IEA GO_REF:0000041 | ACCEPT | Summary: Pathway-vocabulary mapping that places the enzyme at the last step of CDP-diacylglycerol biosynthesis; correct and consistent with the experimental rows. 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. Supporting Evidence: file:ECOLI/cdsA/cdsA-uniprot.txt diacylglycerol from sn-glycerol 3-phosphate: step 3/3. PMID:6273438 Our results constitute definitive physiological proof that CDP-diglyceride is an obligatory precursor for over 90% of the phosphatidylethanolamine and phosphatidylglycerol in E. coli. |
| GO:0005886 plasma membrane | IDA PMID:15919996 Global topology analysis of the Escherichia coli inner membr... | ACCEPT | Summary: Direct inner-membrane assignment from the genome-wide topology study that also fixed this protein's C-terminus in the periplasm. 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. Supporting Evidence: PMID:15919996 we established the periplasmic or cytoplasmic locations of the C termini for 601 inner membrane proteins file:ECOLI/cdsA/cdsA-uniprot.txt SUBCELLULAR LOCATION: Cell inner membrane |
| GO:0005886 plasma membrane | IDA PMID:30751 Partial purification and properties of CTP:phosphatidic acid... | ACCEPT | 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. 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. Supporting Evidence: PMID:30751 was solubilized from the cell envelope by a differential extraction procedure involving the detergent digitonin PMID:2995359 The purification required the use of different detergents at each step, illustrating the refractory hydrophobic nature of this protein. |
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | ACCEPT | 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. 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. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN000349517 SUPPORTS TRANSFER 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. UniProtKB:P0ABG1 SUPPORTS TRANSFER The target's own direct localisation evidence among the descendant evidences; expected, not circular. Supporting Evidence: PMID:2995359 The pure enzyme displays surface dilution kinetics when assayed in the presence of Triton X-100. file:ECOLI/cdsA/cdsA-uniprot.txt SUBCELLULAR LOCATION: Cell inner membrane |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Mapping of the curated UniProt subcellular location, cell inner membrane, onto the nearest GO term; correct for a Gram-negative bacterium. 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. Supporting Evidence: file:ECOLI/cdsA/cdsA-uniprot.txt SUBCELLULAR LOCATION: Cell inner membrane PMID:15919996 we report a global topology analysis of the Escherichia coli inner membrane proteome |
| GO:0016020 membrane | IEA GO_REF:0000002 | MODIFY | Summary: True but less specific than the evidence supports; refine to the plasma membrane term the gene already carries on four other rows. 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 replacements: plasma membrane Supporting Evidence: file:ECOLI/cdsA/cdsA-uniprot.txt SUBCELLULAR LOCATION: Cell inner membrane PMID:15919996 we established the periplasmic or cytoplasmic locations of the C termini for 601 inner membrane proteins |
| GO:0009247 glycolipid biosynthetic process | IMP PMID:30718729 CdsA is involved in biosynthesis of glycolipid MPIase essent... | NEW | Summary: Proposed addition. Beyond bulk phospholipid, CdsA is required for and contributes the lipid unit of MPIase, the glycolipid that drives membrane protein integration. 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. Supporting Evidence: PMID:30718729 We found that MPIase was hardly detected in both the cdsA and cdsA/ynbB knockouts PMID:30718729 These observations indicate that CdsA is a biosynthetic enzyme for MPIase. PMID:30739787 Under CdsA-depleted conditions, YnbB overproduction restored MPIase expression, but not phospholipid biosynthesis. file:ECOLI/cdsA/cdsA-uniprot.txt of MPIase, a glycolipid essential for membrane protein integration and |
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Download this section (compressed HTML)Q: 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.
Suggested experts: GO cellular component curators
Q: 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?
Suggested experts: GO molecular function curators, bacterial membrane biogenesis researchers
Q: 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?
Suggested experts: GO biological process curators, protein translocation researchers
Q: 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?
Suggested experts: EcoCyc curators, GO Consortium biocurators
Experiment: 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.
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.
Type: reconstitution of purified enzymes in proteoliposomes with radiolabelled substrates
Experiment: 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.
Hypothesis: CdsA requires a divalent metal ion for catalysis, as the EDTA sensitivity observed during purification suggests.
Type: enzyme kinetics with chelator treatment and metal add-back
Experiment: 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.
Hypothesis: The active site of CdsA faces the cytoplasm, as required if cytoplasmic CTP is a substrate.
Type: substituted-cysteine accessibility mapping with complementation assays
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: 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.
OPEN BIOLOGY RESIDUAL_SUBGAP
What is known: 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.
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.
What would resolve it: 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 (the field's own admissions):
Gap: 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.
OPEN BIOLOGY RESIDUAL_SUBGAP
What is known: 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.
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.
What would resolve it: 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 (the field's own admissions):
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