LPXA (At4g29540) encodes the Arabidopsis ortholog of bacterial LpxA, a nuclear-encoded, mitochondrially targeted UDP-N-acetylglucosamine acyltransferase that catalyses the first step of the lipid A precursor pathway, transferring an (R)-3-hydroxymyristoyl chain from acyl carrier protein to the 3-OH of UDP-GlcNAc. The purified enzyme is a homotrimer with the left-handed parallel beta-helix fold and conserved active site of E. coli LpxA, catalyses the same reaction in vitro, and complements an E. coli lpxA deletion, restoring normal lipid A synthesis. A GFP fusion localises to mitochondria. Null mutants have no visible phenotype but contain reduced amounts of the downstream intermediate lipid X (2,3-diacylglucosamine 1-phosphate); residual lipid X suggests a second UDP-GlcNAc acylation route. It is one of six enzymes (LpxA, LpxC, LpxD, LpxB, LpxK, KdtA) of a plant pathway that mirrors the E. coli Raetz pathway. The final plant lipid A-like product and its physiological role are unknown.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IEA GO_REF:0000117 | MODIFY | Summary: Cytoplasm, from an ARBA machine-learning rule. Reason: True in the GO sense (mitochondria are part of the cytoplasm) but less specific than the evidence supports. AtLpxA-GFP colocalises with MitoTracker and the protein has an N-terminal mitochondrial transit peptide, so the informative term is mitochondrion (GO:0005739), already carried by the gene from IDA, HDA, IBA and IEA rows. Proposed replacements: mitochondrion Supporting Evidence: PMID:21709257 The GFP fluorescence from the full-length GFP fusions of AtLpxA and AtLpxC1.1 localized in mitochondria |
| GO:0005737 cytoplasm | ISM GO_REF:0000122 | MODIFY | Summary: Cytoplasm, from AtSubP sequence-based prediction (TAIR). Reason: Same claim as the ARBA row. Correct but general; the experimentally supported location is mitochondrion (GO:0005739). Proposed replacements: mitochondrion Supporting Evidence: PMID:21709257 The GFP fluorescence from the full-length GFP fusions of AtLpxA and AtLpxC1.1 localized in mitochondria |
| GO:0005739 mitochondrion | HDA PMID:21908688 In-depth temporal transcriptome profiling reveals a crucial ... | ACCEPT | Summary: Mitochondrion from a germination transcriptome study that also confirmed organellar localisation of a set of proteins by GFP. Reason: The cached abstract (full text not cached) reports GFP confirmation of organellar localisation for 65 proteins but does not name At4g29540; deferring to the curator, who read the full text. The claim agrees with the independent AtLpxA-GFP/MitoTracker colocalisation of Li et al. 2011 and the predicted mitochondrial transit peptide. Supporting Evidence: PMID:21908688 Green fluorescent protein analysis also confirmed organellar localization for 65 proteins PMID:21709257 The GFP fluorescence from the full-length GFP fusions of AtLpxA and AtLpxC1.1 localized in mitochondria |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | ACCEPT | Summary: Mitochondrion propagated by PAINT from node PTN001158739, a plant LpxA clade node whose experimental grounding includes AtLpxA itself. Reason: The node is a plant-specific subfamily (mitochondrial LpxA), and the target appearing among the sources reflects its own IDA localisation, which is expected, not circular. Consistent with GFP data and the transit peptide. Supporting Evidence: PMID:21709257 The GFP fluorescence from the full-length GFP fusions of AtLpxA and AtLpxC1.1 localized in mitochondria |
| GO:0005739 mitochondrion | IDA PMID:21709257 Pathway for lipid A biosynthesis in Arabidopsis thaliana res... | ACCEPT | Summary: Mitochondrion by AtLpxA-GFP fusion colocalisation with MitoTracker in root cells of transgenic Arabidopsis (Li et al. 2011). Reason: Direct experimental localisation using the full-length coding sequence fused to GFP, confirmed by simultaneous MitoTracker staining. Lipid X in atlpxb-1 is also enriched in the mitochondrial fraction. Sub-mitochondrial location is not established, so mitochondrion is the right level of precision. Supporting Evidence: PMID:21709257 The GFP fluorescence from the full-length GFP fusions of AtLpxA and AtLpxC1.1 localized in mitochondria PMID:21709257 Lipid X levels in mitochondria were 3- and 48-fold higher than in chloroplasts or whole cell homogenates, respectively. |
| GO:0005739 mitochondrion | IEA GO_REF:0000044 | ACCEPT | Summary: Mitochondrion from the UniProt subcellular location mapping; the UniProt line is backed by Li et al. 2011 (ECO:0000269). Reason: Same claim as the IDA row; supported by GFP localisation. Supporting Evidence: file:ARATH/LPXA/LPXA-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion {ECO:0000269|PubMed:21709257} PMID:21709257 The GFP fluorescence from the full-length GFP fusions of AtLpxA and AtLpxC1.1 localized in mitochondria |
| GO:0008610 lipid biosynthetic process | IEA GO_REF:0000002 | MODIFY | Summary: Lipid biosynthetic process from InterPro2GO mapping of IPR010137 (Lipid_A_LpxA). Reason: Correct but general. IPR010137 is the LpxA family itself, and AtLpxA catalyses the first step of lipid A precursor synthesis in vitro and restores lipid A synthesis in an E. coli lpxA deletion, so the specific supported process is lipid A biosynthetic process (GO:0009245), proposed here as a NEW experimental annotation. Proposed replacements: lipid A biosynthetic process Supporting Evidence: PMID:22545860 AtLpxA was able to functionally replace EcLpxA by supporting the biosynthesis of the expected lipid A anchor of LPS PMID:21709257 The level of lipid X in the atlpxa-1 insertion mutant was approximately 21% of its matched wild-type control |
| GO:0008780 acyl-[acyl-carrier-protein]-UDP-N-acetylglucosamine O-acyltransferase activity | IBA GO_REF:0000033 | ACCEPT | Summary: LpxA activity propagated by PAINT from node PTN000602373, seeded by E. coli LpxA (P0A722) and by AtLpxA itself. Reason: Core molecular function. AtLpxA is a full-length ortholog of E. coli LpxA with conserved active site residues, and its own in vitro activity (Joo et al. 2012) is part of the descendant evidence for the node; the IBA additionally says the function is inherited across the clade. Supporting Evidence: PMID:22545860 In vitro assays show AtLpxA transfers a 3-hydroxymyristoyl group from 3-hydroxymyristoyl-ACP to the 3-OH of UDP-GlcNAc PMID:21709257 they share 38 and 37% sequence identity with E. coli LpxA, respectively, ( Table 1 ), with conservation of all active site residues |
| GO:0008780 acyl-[acyl-carrier-protein]-UDP-N-acetylglucosamine O-acyltransferase activity | IDA PMID:22545860 Activity and crystal structure of Arabidopsis thaliana UDP-N... | ACCEPT | Summary: LpxA activity shown directly with purified recombinant AtLpxA (Joo et al. 2012). Reason: Purified AtLpxA transfers R-3-hydroxymyristoyl from ACP to UDP-GlcNAc in vitro (specific activity about one tenth of EcLpxA, comparable to other heterologously expressed LpxAs), and AtLpxA complements a chromosomal E. coli lpxA deletion. The crystal structure shows the conserved active site and hydrocarbon ruler residue. Supporting Evidence: PMID:22545860 In vitro assays show AtLpxA transfers a 3-hydroxymyristoyl group from 3-hydroxymyristoyl-ACP to the 3-OH of UDP-GlcNAc PMID:22545860 we have firmly established that the AtLpxA is a functional LpxA through in vitro and in vivo analyses of its biochemical activity |
| GO:0008780 acyl-[acyl-carrier-protein]-UDP-N-acetylglucosamine O-acyltransferase activity | IEA GO_REF:0000120 | ACCEPT | Summary: LpxA activity from combined InterPro/Rhea/EC mapping. Reason: Same claim as the IDA row; the UniProt catalytic activity (RHEA:67812, EC 2.3.1.129) is backed by Joo et al. 2012. Supporting Evidence: PMID:22545860 In vitro assays show AtLpxA transfers a 3-hydroxymyristoyl group from 3-hydroxymyristoyl-ACP to the 3-OH of UDP-GlcNAc |
| GO:0009507 chloroplast | ISM GO_REF:0000122 | REMOVE | Summary: Chloroplast predicted by AtSubP sequence analysis. Reason: Contradicted by experiment. AtLpxA-GFP signal in mesophyll cells formed punctate structures that did not colocalise with chloroplasts, and in roots it colocalised with MitoTracker. The N-terminal presequence is a mitochondrial transit peptide. The only chloroplast link is that some lipid X (in atlpxb-1) is found in chloroplasts, which concerns the lipid, not the enzyme. Supporting Evidence: PMID:21709257 In mesophyll cells, GFP signals were shown as 1 ΞΌm dots, but were not colocalized with chloroplasts PMID:21709257 The GFP fluorescence from the full-length GFP fusions of AtLpxA and AtLpxC1.1 localized in mitochondria |
| GO:2001289 obsolete lipid X metabolic process | IBA GO_REF:0000033 | MODIFY | Summary: Obsolete term "lipid X metabolic process" propagated from plant LpxA node PTN001158739. Reason: QuickGO marks GO:2001289 obsolete ("made in error and is an unnecessary grouping term") with no replacement. The biology behind the row is sound, since LpxA catalyses the first step toward lipid X, and the appropriate current term is lipid A biosynthetic process (GO:0009245), in which LpxA performs the first acyl-transfer step. This is also the term Wei et al. 2020 flagged as a plant misannotation; that flag is refuted for LpxA by direct enzymology and complementation. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN001158739 SUPPORTS TRANSFER Plant mitochondrial LpxA clade node; the inherited role in the first step of lipid A precursor synthesis is sound, but the node carries an obsolete grouping term instead of lipid A biosynthetic process. AGI_LocusCode:AT4G29540 SUPPORTS TRANSFER The target's own IMP (reduced lipid X in atlpxa-1) is descendant evidence for the node, as expected; it supports the biology but under the obsolete term. Proposed replacements: lipid A biosynthetic process Supporting Evidence: PMID:22545860 In vitro assays show AtLpxA transfers a 3-hydroxymyristoyl group from 3-hydroxymyristoyl-ACP to the 3-OH of UDP-GlcNAc PMID:22545860 AtLpxA was able to functionally replace EcLpxA by supporting the biosynthesis of the expected lipid A anchor of LPS PMID:21709257 The level of lipid X in the atlpxa-1 insertion mutant was approximately 21% of its matched wild-type control |
| GO:2001289 obsolete lipid X metabolic process | IMP PMID:21709257 Pathway for lipid A biosynthesis in Arabidopsis thaliana res... | MODIFY | Summary: Obsolete term "lipid X metabolic process" from reduced lipid X in the atlpxa-1 null mutant (Li et al. 2011). Reason: The experiment is sound (lipid X falls to about 21% of wild type in atlpxa-1), but GO:2001289 is obsolete without replacement. The finding supports lipid A biosynthetic process (GO:0009245), the pathway whose first step AtLpxA catalyses. Residual lipid X suggests a second UDP-GlcNAc acylation route, so AtLpxA is the main, not the sole, source. Proposed replacements: lipid A biosynthetic process Supporting Evidence: PMID:21709257 The level of lipid X in the atlpxa-1 insertion mutant was approximately 21% of its matched wild-type control PMID:21709257 the genes encoding AtLpxA, AtLpxC, and AtLpxD2 are required for the efficient production of lipid X |
| GO:0009245 lipid A biosynthetic process | IDA PMID:22545860 Activity and crystal structure of Arabidopsis thaliana UDP-N... | NEW | Summary: NEW. Lipid A biosynthetic process, supported by direct enzymology of AtLpxA (first step of the pathway) and by restoration of lipid A synthesis in an E. coli lpxA deletion. Reason: AtLpxA performs the first step of lipid A biosynthesis itself (catalysis demonstrated in vitro), so it passes the participation test; the comparator check is satisfied because the term's canonical members are exactly the pathway enzymes (E. coli LpxA, LpxC, LpxD, LpxB, LpxK, KdtA). Expressed in an E. coli lpxA knockout, AtLpxA supports synthesis of normal lipid A, and in planta its loss lowers the pathway intermediate lipid X. GOA currently has no lipid A biosynthetic process row for this gene (only the obsolete lipid X term). The plant end product is unknown, but the precursor steps are shared with the E. coli pathway, and the Wei et al. 2020 misannotation flag on plant lipid A terms is refuted by this evidence. Supporting Evidence: PMID:22545860 In vitro assays show AtLpxA transfers a 3-hydroxymyristoyl group from 3-hydroxymyristoyl-ACP to the 3-OH of UDP-GlcNAc PMID:22545860 AtLpxA was able to functionally replace EcLpxA by supporting the biosynthesis of the expected lipid A anchor of LPS PMID:21709257 The level of lipid X in the atlpxa-1 insertion mutant was approximately 21% of its matched wild-type control PMID:21709257 our work demonstrates that plants synthesize lipid A precursors using the same enzymatic pathway present in E. coli |
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Download this section (compressed HTML)Q: Which enzyme accounts for the roughly 21% residual lipid X in atlpxa null mutants; does AtLpxD1, which lacks key LpxD residues, have UDP-GlcNAc acyltransferase activity?
Q: What is the acyl donor for AtLpxA in mitochondria, given that mitochondrial fatty acid synthase is the likely source of (R)-3-hydroxymyristoyl-ACP?
Q: Is the plant enzyme's tenfold lower in vitro specific activity intrinsic, or a consequence of heterologous expression or non-native acyl-ACP?
Experiment: Construct atlpxa atlpxd1 double mutants and quantify lipid X by LC-MRM; assay recombinant AtLpxD1 with UDP-GlcNAc and R-3-hydroxymyristoyl-ACP.
Hypothesis: AtLpxD1 provides the residual UDP-GlcNAc acylation activity in atlpxa mutants.
Type: double mutant lipid profiling
Experiment: Assay AtLpxA with Arabidopsis mitochondrial ACP charged with R-3-hydroxyacyl chains of varying length to define donor and chain-length specificity.
Hypothesis: AtLpxA uses mitochondrial ACP-bound R-3-hydroxyacyl chains as its native donor.
Type: in vitro enzyme assay with plant ACP
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