gpsA (locus PP_4169) encodes the biosynthetic glycerol-3-phosphate dehydrogenase [NAD(P)+] (EC 1.1.1.94) of Pseudomonas putida KT2440. The cytoplasmic enzyme catalyzes the NAD(P)H-dependent reduction of the glycolytic intermediate dihydroxyacetone phosphate (glycerone phosphate, DHAP) to sn-glycerol 3-phosphate (G3P). G3P is the committed precursor for glycerophospholipid (membrane) biosynthesis, making GpsA the entry point that links central carbon metabolism to phospholipid synthesis. The enzyme belongs to the NAD-dependent glycerol-3-phosphate dehydrogenase family, with an N-terminal NAD(P)-binding Rossmann-like domain, and can use either NAD(H) or NADP(H) as the redox cofactor. In vivo the physiologically relevant flux is in the biosynthetic (reductive) DHAP-to-G3P direction.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005737 cytoplasm | IEA GO_REF:0000120 | ACCEPT | Summary: Cytoplasmic localization is directly supported by the UniProt subcellular-location statement for this soluble dehydrogenase. Reason: GpsA is a soluble cytoplasmic enzyme; the UniProt record explicitly assigns it to the cytoplasm. Supporting Evidence: file:PSEPK/gpsA/gpsA-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0005829 cytosol | IEA GO_REF:0000118 | KEEP AS NON CORE | Summary: Cytosol is compatible with the cytoplasmic location but is a redundant, more specific compartment term and is not the core function. Reason: Consistent with the UniProt cytoplasm assignment; retained as a non-core location annotation. Supporting Evidence: file:PSEPK/gpsA/gpsA-uniprot.txt SUBCELLULAR LOCATION: Cytoplasm |
| GO:0005975 carbohydrate metabolic process | IEA GO_REF:0000002 | MODIFY | Summary: This InterPro-based transfer is misleading. GpsA is a glycerophospholipid-precursor enzyme; its substrate DHAP is a glycolytic intermediate, but the enzyme does not function in carbohydrate metabolism. The biosynthetic process terms capture its true role. Reason: The enzyme reduces DHAP to glycerol-3-phosphate to feed phospholipid synthesis, not carbohydrate metabolism; replace with the specific glycerophospholipid biosynthetic process term. Proposed replacements: glycerophospholipid biosynthetic process Supporting Evidence: file:PSEPK/gpsA/gpsA-uniprot.txt key precursor for phospholipid synthesis. |
| GO:0006072 glycerol-3-phosphate metabolic process | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Correct but broad. The enzyme participates in glycerol-3-phosphate metabolism, but its specific role is the biosynthetic (production) branch. Reason: A correct parent process; the more specific biosynthetic term is preferred for core annotation. Supporting Evidence: file:PSEPK/gpsA/gpsA-uniprot.txt dihydroxyacetone phosphate (DHAP) to sn-glycerol 3-phosphate (G3P), the |
| GO:0006650 glycerophospholipid metabolic process | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: Correct but broad. GpsA supplies the G3P precursor for glycerophospholipid synthesis; the biosynthetic child term is more informative. Reason: A correct parent process; the specific glycerophospholipid biosynthetic process term better represents the gene's role. Supporting Evidence: file:PSEPK/gpsA/gpsA-uniprot.txt PATHWAY: Membrane lipid metabolism; glycerophospholipid metabolism. |
| GO:0016616 oxidoreductase activity, acting on the CH-OH group of donors, NAD or NADP as acceptor | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: True but broad. This is a parent of the specific glycerol-3-phosphate dehydrogenase [NAD(P)+] activity, which is annotated; this general term is an over-annotation. Reason: The specific activity term GO:0047952 is present and captures the function more precisely; the general oxidoreductase class adds no information. Supporting Evidence: file:PSEPK/gpsA/gpsA-uniprot.txt sn-glycerol 3-phosphate + NAD(+) = dihydroxyacetone phosphate |
| GO:0046167 glycerol-3-phosphate biosynthetic process | IEA GO_REF:0000104 | ACCEPT | Summary: Accurate core process. GpsA produces sn-glycerol 3-phosphate from DHAP, the defining biosynthetic step. Reason: Directly supported by the UniProt function statement describing reduction of DHAP to G3P. Supporting Evidence: file:PSEPK/gpsA/gpsA-uniprot.txt dihydroxyacetone phosphate (DHAP) to sn-glycerol 3-phosphate (G3P), the |
| GO:0046168 glycerol-3-phosphate catabolic process | IEA GO_REF:0000002 | REMOVE | Summary: Incorrect for this gene. gpsA is the biosynthetic isozyme that produces G3P; it does not catabolize it. G3P catabolism is carried out by the distinct respiratory GlpD/GlpABC dehydrogenases. This is a family-level InterPro transfer that conflates the two directions. Reason: The biosynthetic GpsA produces rather than degrades glycerol-3-phosphate; the catabolic annotation is the wrong direction for this gene. PANTHER classifies gpsA in the biosynthetic NAD(P)+ glycerol-3-phosphate dehydrogenase family/subfamily (PTHR11728 / PTHR11728:SF1), despite the eukaryote-biased ", CHLOROPLASTIC" subfamily naming artifact, reinforcing the G3P-producing rather than G3P-catabolizing assignment. Supporting Evidence: file:PSEPK/gpsA/gpsA-uniprot.txt key precursor for phospholipid synthesis. file:PSEPK/gpsA/gpsA-notes.md this biosynthetic isozyme (gpsA) produces G3P file:PSEPK/gpsA/gpsA-uniprot.txt PTHR11728:SF1; GLYCEROL-3-PHOSPHATE DEHYDROGENASE [NAD(+)] 2, CHLOROPLASTIC |
| GO:0046474 glycerophospholipid biosynthetic process | IEA GO_REF:0000118 | ACCEPT | Summary: Accurate core process. By producing G3P, the committed precursor, GpsA feeds glycerophospholipid (membrane) biosynthesis. Reason: G3P is described as the key precursor for phospholipid synthesis, and the UniPathway assignment is glycerophospholipid metabolism. Supporting Evidence: file:PSEPK/gpsA/gpsA-uniprot.txt key precursor for phospholipid synthesis. |
| GO:0047952 glycerol-3-phosphate dehydrogenase [NAD(P)+] activity | IEA GO_REF:0000120 | ACCEPT | Summary: Correct core molecular function (EC 1.1.1.94). This is the precise activity of GpsA, using NAD(H) or NADP(H). Reason: Directly matches the UniProt RecName and the dual NAD(+)/NADP(+) catalytic-activity reactions (EC 1.1.1.94). Supporting Evidence: file:PSEPK/gpsA/gpsA-uniprot.txt EC=1.1.1.94 {ECO:0000255|HAMAP-Rule:MF_00394}; file:PSEPK/gpsA/gpsA-uniprot.txt sn-glycerol 3-phosphate + NADP(+) = dihydroxyacetone phosphate file:PSEPK/gpsA/gpsA-uniprot.txt PTHR11728; GLYCEROL-3-PHOSPHATE DEHYDROGENASE |
| GO:0051287 NAD binding | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: The enzyme binds NAD(P) cofactor via its Rossmann-like domain, but this broad cofactor-binding term is over-annotated relative to the specific catalytic activity, and it under-represents the NADP(H) usage. Reason: Cofactor binding is implied by the specific dehydrogenase activity; the generic NAD binding term adds little and omits the NADP specificity of this enzyme. Supporting Evidence: file:PSEPK/gpsA/gpsA-uniprot.txt Belongs to the NAD-dependent glycerol-3-phosphate |
| GO:0141152 glycerol-3-phosphate dehydrogenase (NAD+) activity | IEA GO_REF:0000116 | ACCEPT | Summary: Accurate Rhea-derived sub-activity. The enzyme catalyzes the NAD(+)-linked reaction (RHEA:11092). Reason: The UniProt catalytic-activity block records the NAD(+) reaction mapped to RHEA:11092. Supporting Evidence: file:PSEPK/gpsA/gpsA-uniprot.txt sn-glycerol 3-phosphate + NAD(+) = dihydroxyacetone phosphate |
| GO:0141153 glycerol-3-phosphate dehydrogenase (NADP+) activity | IEA GO_REF:0000116 | ACCEPT | Summary: Accurate Rhea-derived sub-activity. The enzyme also catalyzes the NADP(+)-linked reaction (RHEA:11096), reflecting its dual cofactor specificity. Reason: The UniProt catalytic-activity block records the NADP(+) reaction mapped to RHEA:11096. Supporting Evidence: file:PSEPK/gpsA/gpsA-uniprot.txt sn-glycerol 3-phosphate + NADP(+) = dihydroxyacetone phosphate |
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Download this section (compressed HTML)Q: Is GpsA the sole route to sn-glycerol 3-phosphate for phospholipid biosynthesis in P. putida KT2440, or can G3P also be supplied by glycerol kinase (GlpK) under glycerol-rich conditions?
Suggested experts: Pseudomonas lipid metabolism experts
Q: What is the in vivo cofactor preference (NADH vs NADPH) of P. putida GpsA, and how does it integrate with the cell's redox/NADPH balance during membrane lipid synthesis?
Suggested experts: Bacterial enzymology experts
Experiment: Express and purify recombinant P. putida GpsA and measure steady-state kinetics for DHAP reduction with NADH and with NADPH to quantify dual cofactor specificity and the preferred physiological direction.
Type: enzyme kinetics assay
Experiment: Construct a gpsA deletion in KT2440 and test for glycerol-3-phosphate auxotrophy and membrane phospholipid defects, with complementation to confirm the biosynthetic role.
Type: gene knockout / complementation
Experiment: Use isotopically labeled glucose/DHAP and lipidomics to trace flux through GpsA into sn-glycerol 3-phosphate and downstream glycerophospholipids in KT2440.
Type: metabolite tracing
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