EstA (PA5112) is an outer-membrane autotransporter esterase of Pseudomonas aeruginosa. Its N-terminal GDSL/SGNH-family passenger domain, with a Ser-Asp-His catalytic triad, is translocated across the outer membrane by the C-terminal 12-stranded beta-barrel domain and remains covalently attached, exposing the active site on the cell surface. The enzyme is a carboxylesterase that prefers short-chain acyl esters (p-nitrophenyl butyrate to hexanoate), Tween detergents and beta-naphthyl butyrate, with negligible activity on triacylglycerols and acyl-CoA. Deletion of estA strongly reduces extracellular rhamnolipid and impairs swimming, swarming and twitching motility and biofilm formation, and these phenotypes require the catalytically active enzyme; the physiological substrate and the step by which EstA influences rhamnolipid levels are unknown. EstA is also widely used as an anchor for bacterial cell-surface display.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0006629 lipid metabolic process | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: Family-level InterPro mapping (GDSL lipase active site) to lipid metabolism; no lipid substrate of EstA has been established. Reason: IPR008265 marks the GDSL serine active-site motif shared by lipases and esterases. EstA is a short-chain carboxylesterase with negligible activity on triacylglycerols and acyl-CoA, and in a 34-substrate panel only the Streptomyces enzyme, not EstA, showed lipase or phospholipase activity. Its requirement for rhamnolipid production is a phenotype of unknown mechanism. The mapping therefore asserts lipid metabolism without evidence that EstA acts on a lipid in vivo. Supporting Evidence: file:PSEAE/estA/estA-uniprot.txt acyls-CoA are negligible PMID:20931591 Of all the enzymes tested, only SrLip revealed lipase, phospholipase, esterase, thioesterase and Tweenase activities |
| GO:0008610 lipid biosynthetic process | IBA GO_REF:0000033 | MARK AS OVER ANNOTATED | Summary: IBA from the PTN005219546 IBD whose only seed is EstA itself; the seed rests on a rhamnolipid-deficient mutant phenotype, not on participation in lipid synthesis. Reason: The IBD at PTN005219546 for lipid biosynthetic process is sole-seeded by EstA (UniProt IMP GO:0008610 and PseudoCAP IMP GO:0009247, both PMID:17631636) and projects to 960 leaves in 51 taxa. The underlying evidence is that an estA deletion makes little rhamnolipid and that an active-site Ser->Ala variant does not complement. That establishes necessity of the esterase activity, not that EstA performs a biosynthetic step; rhamnolipid is assembled by RhlA, RhlB and RhlC, and EstA is a surface-exposed outer-membrane esterase with negligible activity on acyl-CoA. UniProt itself states that the role of EstA in these processes is unclear. No other member of the GDSL autotransporter esterase clade has evidence for lipid biosynthesis. On EstA the row is kept only as an over-annotated restatement of the phenotype; the node annotation should be withdrawn (see source_defects). Propagation Review Root cause: SOURCE BAD Failure modes: ROLE CONFLATION Sources checked: PANTHER:PTN005219546 · PTN005219546 SOURCE BAD Sole seed is EstA's own IMP phenotype rows (GO:0008610 UniProt, GO:0009247 PseudoCAP, PMID:17631636); a necessity phenotype of unknown mechanism, not participation in lipid biosynthesis. Supporting Evidence: PMID:17631636 In order to demonstrate that EstA enzyme activity is responsible for these effects, inactive variant EstA* was constructed by replacement of the active serine by alanine. file:PSEAE/estA/estA-uniprot.txt the exact role of EstA in these PMID:10559163 indicating that the enzyme was located in the outer membrane with the catalytic domain exposed to the surface |
| GO:0008610 lipid biosynthetic process | IMP PMID:17631636 The autotransporter esterase EstA of Pseudomonas aeruginosa ... | MARK AS OVER ANNOTATED | Summary: Deletion mutant phenotype (marginal rhamnolipid) annotated as participation in lipid biosynthesis; the step EstA affects is unknown. Reason: The abstract establishes that EstA catalytic activity is required for normal rhamnolipid levels and that overexpression increases them. It does not identify a rhamnolipid intermediate as an EstA substrate, and EstA acts at the cell surface, outside the compartment where RhlA/RhlB/RhlC synthesise rhamnolipid. The full text was not available, so I do not remove the row, but as stated it records a necessity phenotype rather than a biosynthetic role. This row is the sole seed of the PTN005219546 lipid biosynthetic process IBD. Supporting Evidence: PMID:17631636 an estA deletion mutant produced only marginal amounts of rhamnolipids PMID:17631636 None of the mutant phenotypes could be complemented by expression of EstA* file:PSEAE/estA/estA-uniprot.txt the exact role of EstA in these |
| GO:0009247 glycolipid biosynthetic process | IMP PMID:17631636 The autotransporter esterase EstA of Pseudomonas aeruginosa ... | MARK AS OVER ANNOTATED | Summary: PseudoCAP IMP from the same rhamnolipid-deficient phenotype; supports the lipid biosynthetic process IBD seed; indirect phenotype. Reason: Rhamnolipid is a glycolipid, so the term names the affected product correctly, but the evidence is a deletion phenotype whose mechanism is unknown (see the GO:0008610 IMP row). EstA has no demonstrated activity on any rhamnolipid precursor and is located on the cell surface. The abstract does not show participation; I mark it over-annotated rather than removing it because the full text could not be read. Supporting Evidence: PMID:17631636 an estA deletion mutant produced only marginal amounts of rhamnolipids file:PSEAE/estA/estA-uniprot.txt the exact role of EstA in these |
| GO:0009279 cell outer membrane | IDA PMID:17631636 The autotransporter esterase EstA of Pseudomonas aeruginosa ... | ACCEPT | Summary: Outer-membrane location with surface-exposed catalytic domain is well established. Reason: EstA is an autotransporter whose beta-barrel inserts in the outer membrane; fractionation and protease accessibility (PMID:10559163), the 2007 study, and the full-length crystal structure all support outer-membrane location with the esterase domain exposed on the surface. Supporting Evidence: PMID:17631636 EstA is an autotransporter protein which exposes its catalytically active esterase domain on the cell surface. PMID:10559163 indicating that the enzyme was located in the outer membrane with the catalytic domain exposed to the surface PMID:20060837 EstA has a relatively narrow, 12-stranded beta-barrel |
| GO:0009279 cell outer membrane | IEA GO_REF:0000044 | ACCEPT | Summary: Mapping of the curated UniProt subcellular location (cell outer membrane). Reason: The UniProt location is experimentally based (PMID:10559163, PMID:17631636), consistent with the IDA row. Supporting Evidence: file:PSEAE/estA/estA-uniprot.txt SUBCELLULAR LOCATION: Cell outer membrane |
| GO:0016298 lipase activity | IEA GO_REF:0000002 | MODIFY | Summary: InterPro GDSL-lipase-active-site mapping; EstA is an esterase with negligible lipase activity. Reason: GDSL family members include true lipases, but EstA prefers short-chain p-nitrophenyl esters, has negligible activity on triacylglycerols, and showed no lipase or phospholipase activity in a 34-substrate panel. The correct activity is carboxylesterase activity, which the gene already carries by IDA. Proposed replacements: carboxylesterase activity Supporting Evidence: PMID:20931591 Of all the enzymes tested, only SrLip revealed lipase, phospholipase, esterase, thioesterase and Tweenase activities file:PSEAE/estA/estA-uniprot.txt acyls-CoA are negligible |
| GO:0016788 hydrolase activity, acting on ester bonds | IEA GO_REF:0000002 | MODIFY | Summary: Correct but general; carboxylesterase activity is experimentally supported. Reason: The GDSL domain mapping is correct, but EstA is shown to hydrolyse carboxylic esters (p-nitrophenyl butyrate, KM 0.7 mM), so the specific term GO:0106435 carboxylesterase activity, already carried by the gene, is preferred. Proposed replacements: carboxylesterase activity Supporting Evidence: file:PSEAE/estA/estA-uniprot.txt KM=0.7 mM for p-nitrophenyl butyrate file:PSEAE/estA/estA-uniprot.txt Pfam; PF00657; Lipase_GDSL; 1. |
| GO:0052689 carboxylic ester hydrolase activity | IBA GO_REF:0000033 | MODIFY | Summary: Sound IBA from the PTN005219546 GDSL esterase node; correct but general for EstA, whose carboxylesterase activity is directly shown. Reason: The IBD is seeded by EstA (IMP and IDA esterase evidence) and by PA4921 (Q9HUP2, IDA choline esterase), and carboxylic ester hydrolysis is the conserved GDSL/SGNH chemistry of the clade, so the node call is sound. EstA appearing in its own WITH/FROM reflects its experimental grounding. For EstA itself the more specific descendant GO:0106435 carboxylesterase activity is supported by IDA and kinetics. Propagation Review Root cause: NO FAILURE CORE Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN005219546 · PTN005219546 SUPPORTS TRANSFER Seeds are EstA (IMP PseudoCAP, IDA UniProt) and PA4921/Q9HUP2 (IDA PseudoCAP choline esterase); node-level carboxylic ester hydrolase is sound. Proposed replacements: carboxylesterase activity Supporting Evidence: PMID:20931591 TesA, EstA and EstP were shown to function as esterases with different affinities and catalytic efficacies towards p-nitrophenyl butyrate file:PSEAE/estA/estA-uniprot.txt Belongs to the 'GDSL' lipolytic enzyme family. |
| GO:0052689 carboxylic ester hydrolase activity | IMP PMID:17631636 The autotransporter esterase EstA of Pseudomonas aeruginosa ... | MODIFY | Summary: Sound PseudoCAP seed; the active-site Ser->Ala variant is catalytically inactive. Correct but general relative to carboxylesterase activity. Reason: The 2007 paper constructed an active-site serine-to-alanine variant to remove esterase activity, a valid IMP for the activity, and esterase activity of EstA is independently shown (PMID:10559163, PMID:20931591). This seed of the PTN005219546 carboxylic ester hydrolase IBD is sound. The supported specific term is GO:0106435 carboxylesterase activity, already on the gene. Proposed replacements: carboxylesterase activity Supporting Evidence: PMID:17631636 inactive variant EstA* was constructed by replacement of the active serine by alanine PMID:10559163 extracellular lipolytic activity toward short-chain p-nitrophenylesters |
| GO:0106435 carboxylesterase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic EC 3.1.1.1 mapping, consistent with experimental data. Reason: EC 3.1.1.1 is assigned in the curated catalytic-activity comment from experimental evidence (PMID:10559163, PMID:20931591); the electronic row agrees with the IDA row. Supporting Evidence: PMID:20931591 TesA, EstA and EstP were shown to function as esterases with different affinities and catalytic efficacies towards p-nitrophenyl butyrate |
| GO:0106435 carboxylesterase activity | IDA PMID:20931591 Probing enzyme promiscuity of SGNH hydrolases. | ACCEPT | Summary: Direct assay of EstA esterase activity and kinetics on p-nitrophenyl butyrate; core function. Reason: EstA was tested against 34 substrates and characterised kinetically as an esterase of short-chain acyl esters (KM 0.7 mM for p-nitrophenyl butyrate). Supporting Evidence: PMID:20931591 TesA, EstA and EstP were shown to function as esterases with different affinities and catalytic efficacies towards p-nitrophenyl butyrate file:PSEAE/estA/estA-uniprot.txt KM=0.7 mM for p-nitrophenyl butyrate |
| GO:0044010 single-species biofilm formation | IMP PMID:17631636 The autotransporter esterase EstA of Pseudomonas aeruginosa ... | KEEP AS NON CORE | Summary: Biofilm formation is affected in the estA mutant; a phenotype-level process, likely secondary to reduced rhamnolipid. Reason: The abstract reports that biofilm formation was affected by estA mutation and that the phenotype needs active enzyme. Biofilm IMP annotations of this kind are standard bacterial practice and the claim is true at the phenotype level, but it is peripheral and probably indirect (via rhamnolipid). Not a seed of any IBD. Supporting Evidence: PMID:17631636 Also the known rhamnolipid-dependent cellular motility and biofilm formation were affected. |
| GO:0071973 bacterial-type flagellum-dependent cell motility | IMP PMID:17631636 The autotransporter esterase EstA of Pseudomonas aeruginosa ... | MARK AS OVER ANNOTATED | Summary: Swimming is affected in the mutant, but so is pilus-driven twitching; the flagellum-specific term asserts a mechanism that is not shown. Reason: The estA mutation affects swimming, swarming and twitching alike, which span flagellar and type IV pilus machineries; a surface esterase is not part of the flagellar motility process, and the effect is most plausibly pleiotropic. The same pleiotropy argument applies to the general cell motility row. Supporting Evidence: PMID:17631636 the other kinds of motility displayed by P. aeruginosa PAO1, swimming and twitching, were also affected by an estA mutation |
| GO:0071978 bacterial-type flagellum-dependent swarming motility | IDA PMID:20360178 Extracellular enzymes affect biofilm formation of mucoid Pse... | MARK AS OVER ANNOTATED | Summary: Based on an estA overexpression phenotype in a mucoid strain (enhanced swarming); indirect, and an overexpression phenotype is IMP, not IDA. Reason: The paper overexpressed estA and observed enhanced swimming and swarming but loss of twitching, and the authors say the enzymes act directly or indirectly through extracellular polymeric substance composition. Swarming depends on rhamnolipid as a surfactant, so the effect is plausibly secondary to rhamnolipid levels. The row overstates EstA as a participant in swarming, and the evidence code should be IMP. Supporting Evidence: PMID:20360178 Overexpression of estA resulted in a loss of twitching motility, although it enhanced the ability to swim and swarm. PMID:20360178 directly or indirectly, the secreted enzymes EstA, LasB and LipC can influence the formation and architecture of mucoid P. aeruginosa biofilms |
| GO:0048870 cell motility | IMP PMID:17631636 The autotransporter esterase EstA of Pseudomonas aeruginosa ... | MARK AS OVER ANNOTATED | Summary: Loss of swimming, swarming and twitching in the estA mutant; a phenotype-level process. Reason: All motility forms are affected in the estA mutant, but involved_in on the general term still asserts that EstA takes part in cell motility, and the argument that marks the flagellar rows applies here too. The effect spans flagellar and type IV pilus machineries, the mechanism is unknown, and it is most plausibly pleiotropic, for example through rhamnolipid or extracellular polymeric substance levels. Over-annotation rather than removal, because the full text was not read. Supporting Evidence: PMID:17631636 the other kinds of motility displayed by P. aeruginosa PAO1, swimming and twitching, were also affected by an estA mutation |
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Download this section (compressed HTML)Q: Should PAINT withdraw the GO:0008610 lipid biosynthetic process IBD at PTN005219546, given that its only seed is a rhamnolipid-deficient estA mutant phenotype?
Q: Does EstA affect rhamnolipid biosynthesis, export, or turnover, and is the effect mediated through rhl quorum-sensing regulation of rhlAB expression?
Experiment: Lipidomic and metabolomic profiling of wild-type, estA deletion, and EstA S38A strains (cell-associated and supernatant fractions), combined with rhlAB reporter assays, to determine whether EstA alters rhamnolipid synthesis, release, or regulation, and to identify candidate in vivo ester substrates.
What is not known — curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: The physiological substrate of EstA and the mechanism by which its esterase activity is required for rhamnolipid accumulation, motility and biofilm formation are unknown; EstA has no activity on acyl-CoA and is located outside the compartment where rhamnolipid is synthesised.
BIOLOGY
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