PlsY (formerly YneS) is the glycerol-phosphate acyltransferase that performs the first acylation step of membrane phospholipid synthesis in Bacillus subtilis. It is a small polytopic integral protein of the cytoplasmic membrane that transfers a fatty acyl group from acyl-phosphate, made from acyl-ACP by PlsX, to sn-glycerol 3-phosphate to form 1-acyl-sn-glycerol 3-phosphate (lysophosphatidic acid); it uses neither acyl-CoA nor acyl-ACP as donor, and is inhibited by acyl-CoA. B. subtilis lacks PlsB, so PlsY is its only glycerol-phosphate acyltransferase and is required for phospholipid synthesis and growth. Purified PlsY also acylates sn-glycerol 1-phosphate in vitro, at levels close to those with glycerol 3-phosphate in detergent but with a clear glycerol 3-phosphate preference when reconstituted in a membrane. That activity could account for the minor glycerol 1-phosphate-backbone phosphatidylglycerol reported in B. subtilis, but its use in the living cell has not been shown.
Definition: Catalysis of the reaction: acyl phosphate + sn-glycerol 1-phosphate = 3-acyl-sn-glycerol 1-phosphate + orthophosphate.
Justification: GO has acyl-phosphate glycerol-3-phosphate acyltransferase activity (GO:0043772) for the PlsX/PlsY reaction on the R enantiomer, and an OLS search of the acyltransferase branch found no counterpart for sn-glycerol 1-phosphate, the S enantiomer that archaeal lipids use. Purified B. subtilis and E. coli PlsY acylate glycerol 1-phosphate as well as glycerol 3-phosphate, so the reaction is directly observed and there is currently no way to state it in GO. The term is requested so that the observation can be recorded when a curator judges it annotatable; this review does not itself assert the activity for B. subtilis PlsY in vivo, because the enzyme prefers glycerol 3-phosphate in a membrane, no kinetic constants exist, the in vivo source of glycerol 1-phosphate is unidentified, and the heterochiral lipid report that motivates the inference comes from a single laboratory. The companion reaction on the product side, acylation of (S)-lysophosphatidic acid by PlsC, has the same status and would be a separate request.
Parent term: acyltransferase activity, transferring groups other than amino-acyl groups
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | ACCEPT | Summary: Plasma membrane location of the PlsY family, propagated from E. coli PlsY; correct for B. subtilis PlsY. Reason: PlsY is a polytopic membrane protein with five predicted transmembrane helices, and acyl-phosphate-dependent G3P acylation activity resides in the B. subtilis membrane fraction and is absent from membranes of the deletion strain. B. subtilis is a monoderm, so its only membrane is the cytoplasmic (plasma) membrane, where phospholipid synthesis takes place. The PAINT node is seeded by E. coli PlsY (P60782); B. subtilis PlsY belongs to the same PlsY family and nothing suggests a divergent location, so the node placement holds. A single seed is not a weakness for a family whose members are all integral membrane proteins. Supporting Evidence: PMID:19282621 Membrane fractions from B. subtilis cells catalyzed palmitoylphosphate-dependent acylation of [14C]-labeled G3P to synthesize [14C]-labeled LPA, whereas those from DeltaplsY cells did not. file:BACSU/plsY/plsY-uniprot.txt TRANSMEM 2..22 PMID:42286214 Although PlsY is located in the membrane, its activity levels in detergent are comparable (Lu et al, 2007). |
| GO:0005886 plasma membrane | IEA GO_REF:0000120 | ACCEPT | Summary: Plasma membrane location from the PlsY family signature and the UniProt subcellular location vocabulary; correct. Reason: Same claim as the IBA row, and consistent with it. Five predicted transmembrane helices and membrane-fraction activity that depends on plsY place PlsY in the cytoplasmic membrane of this monoderm bacterium. Supporting Evidence: PMID:17645809 the plsY gene, encoding a membrane protein around 200 amino acids long, is also widely conserved in eubacteria PMID:19282621 Membrane fractions from B. subtilis cells catalyzed palmitoylphosphate-dependent acylation of [14C]-labeled G3P to synthesize [14C]-labeled LPA, whereas those from DeltaplsY cells did not. |
| GO:0006644 phospholipid metabolic process | IEA GO_REF:0000041 | MODIFY | Summary: True but uninformative; the specific biosynthetic step PlsY catalyses is known. Reason: The claim is correct, but the evidence supports a far more precise descendant. PlsY catalyses the first of the two acylations that make phosphatidic acid, and loss of PlsY abolishes phospholipid synthesis in vivo, so phosphatidic acid biosynthetic process (GO:0006654), a descendant of this term, is the supported granularity. This is a refinement, not a correction. Proposed replacements: phosphatidic acid biosynthetic process Supporting Evidence: PMID:17557823 plsX (acyl-acyl carrier protein [ACP]:phosphate acyltransferase), plsY (yneS) (acyl-phosphate:glycerol-phosphate acyltransferase), and plsC (yhdO) (acyl-ACP:1-acylglycerol-phosphate acyltransferase) function in phosphatidic acid formation, the precursor to membrane phospholipids. PMID:17557823 The inactivation of PlsY also blocked phospholipid synthesis |
| GO:0008654 phospholipid biosynthetic process | IEA GO_REF:0000120 | MODIFY | Summary: Correct, but less specific than the characterized step; refine to phosphatidic acid biosynthesis. Reason: PlsY is required for all phospholipid synthesis in B. subtilis, so the term is true. The step it performs is the sn-1 acylation of G3P en route to phosphatidic acid, and the temperature-sensitive and depletion phenotypes pin the block to that step, so GO:0006654 is the supported descendant. Consistent with the MODIFY on the GO:0006644 row. Proposed replacements: phosphatidic acid biosynthetic process Supporting Evidence: PMID:17645809 no phospholipids were detectable in yneS-ts cells following a shift to the restrictive temperature PMID:42286214 This process starts with the attachment of a first activated fatty acid to the primary carbon of a glycerol-phosphate through an esterification, which is catalyzed by enzymes belonging to the family of glycerol-phosphate acyltransferases (GPAT) |
| GO:0043772 acyl-phosphate glycerol-3-phosphate acyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: The core catalytic activity of PlsY, established experimentally in B. subtilis itself. Reason: The electronic assignment matches direct evidence on this gene. B. subtilis membranes carry out palmitoyl-phosphate-dependent acylation of G3P to LPA, and that activity is lost in a plsY deletion; the donor specificity is acyl-phosphate, not acyl-CoA or acyl-ACP. The term's reaction is exactly the characterized one (Rhea:34075, EC 2.3.1.275). Supporting Evidence: PMID:19282621 The results indicate unequivocally that PlsY is an acyl-phosphate dependent GPAT. PMID:17557823 PlsY was an acylphosphate-specific acyltransferase file:BACSU/plsY/plsY-uniprot.txt Catalyzes the transfer of an acyl group from acyl-phosphate |
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Download this section (compressed HTML)Q: Should GO provide stereochemistry-neutral parent terms for the glycerol-phosphate acylation reactions, with the G3P and G1P reactions as children, rather than only the G3P-specific terms it has now?
Suggested experts: GO molecular function curators, lipid metabolism curators
Q: Given that PlsY is the sole GPAT of B. subtilis and that its phosphatidylglycerol carries both backbone enantiomers, what evidence would GO require before annotating a stereo-alternative activity to a bacterial acyltransferase?
Suggested experts: GO Consortium biocurators, bacterial lipid biochemists
Q: Is the acyl-phosphate route the point at which the glycerol-phosphate enantiomer is selected, or is selection made upstream by the dehydrogenases that supply G3P and G1P?
Suggested experts: Bacterial membrane lipid biochemists
Experiment: Deplete PlsY in cells complemented by E. coli plsB, which is strictly specific for glycerol 3-phosphate, and analyse membrane phospholipid backbone chirality by chiral chromatography-mass spectrometry. Loss of the S-backbone species on PlsB-only complementation would show the activity is PlsY's; its persistence would point to another enzyme or to a headgroup artefact.
Hypothesis: The minor glycerol 1-phosphate-backbone phosphatidylglycerol of B. subtilis is made by PlsY acting on glycerol 1-phosphate.
Type: genetic complementation plus chiral lipidomics
Experiment: Compare backbone chirality and glycerol 1-phosphate pools in wild type, an araM (egsA) deletion, and a strain overexpressing araM, by GC/MS and LC/MS of glycerol phosphates and chiral analysis of phosphatidylglycerol.
Hypothesis: Glycerol 1-phosphate supply, not PlsY specificity, limits heterochiral lipid production.
Type: metabolite and lipid quantification in defined mutants
Experiment: Determine steady-state kinetic constants of reconstituted B. subtilis PlsY for glycerol 3-phosphate and glycerol 1-phosphate in a single-enzyme assay with chemically synthesised acyl-phosphate, in detergent and in liposomes, so the coupled-assay limitation of the published work is removed.
Hypothesis: PlsY discriminates between the glycerol-phosphate enantiomers kinetically, with a preference that depends on the membrane environment.
Type: enzyme kinetics on reconstituted protein
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: Whether PlsY acylates sn-glycerol 1-phosphate in the living cell, and so whether it has a second, stereochemically distinct physiological reaction, is unknown.
OPEN BIOLOGY RESIDUAL_SUBGAP
What is known: What is established is that purified B. subtilis PlsY acylates glycerol 1-phosphate as well as glycerol 3-phosphate in detergent, with only minor differences in product levels, and that in liposomes it still acylates glycerol 1-phosphate but clearly prefers glycerol 3-phosphate; separately, that B. subtilis phosphatidylglycerol contains both glycerol-phosphate enantiomers. What is unknown is whether the glycerol 1-phosphate-backbone lipid is made by PlsY, what supplies its glycerol 1-phosphate in vivo, and what the kinetic preference is.
Significance: B. subtilis is the test case for the lipid divide being crossable within ordinary bacterial phospholipid biosynthesis. If PlsY acylates glycerol 1-phosphate in vivo, one bacterial enzyme accounts for heterochiral membranes, and GO currently has no term for that reaction.
What would resolve it: Measure the dependence of glycerol 1-phosphate-backbone phosphatidylglycerol on PlsY and on glycerol 1-phosphate supply, by chiral lipid analysis of an araM (egsA) deletion and of PlsY-depleted cells complemented with the strictly glycerol 3-phosphate-specific E. coli PlsB, and determine kinetic constants for both enantiomers on reconstituted PlsY.
Provenance (the field's own admissions):
Gap: The source of sn-glycerol 1-phosphate in B. subtilis, and whether its supply rather than acyltransferase specificity sets the fraction of heterochiral lipid, is unresolved.
OPEN BIOLOGY BP_DARK
What is known: AraM (EgsA) is the only known bacterial glycerol-1-phosphate dehydrogenase, but engineered E. coli made correctly configured archaeal lipids without AraM, so at least one other route to glycerol 1-phosphate exists in bacteria and is unidentified.
Significance: If glycerol 1-phosphate supply is limiting, the stereochemistry of the bacterial membrane is set upstream of PlsY, and the acyltransferases are permissive rather than determining; that distinction decides whether a stereo-specific molecular function term belongs on PlsY at all.
What would resolve it: Quantify glycerol 1-phosphate pools and lipid backbone chirality in wild-type and araM deletion B. subtilis, and search genetically for the alternative route suggested by the engineered E. coli result.
Provenance (the field's own admissions):
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