WaaC (RfaC) is the lipopolysaccharide heptosyltransferase I of Pseudomonas aeruginosa, a 355-residue glycosyltransferase family 9 enzyme that transfers the first L-glycero-D-manno-heptose from ADP-heptose to the Kdo2-lipid A acceptor, starting assembly of the inner core oligosaccharide. It is a peripheral protein on the cytoplasmic face of the inner membrane, where the lipid A acceptor resides. The PAO1 gene restores smooth LPS to a Salmonella waaC mutant, and chromosomal null mutants could not be isolated in P. aeruginosa, consistent with the inner core being required for viability or outer membrane integrity in this organism. waaC lies next to waaF, which adds the second heptose.
Definition: Catalysis of the reaction: an alpha-Kdo-(2->4)-alpha-Kdo-(2->6)-lipid A + ADP-L-glycero-beta-D-manno-heptose = an L-alpha-D-Hep-(1->5)-[alpha-Kdo-(2->4)]-alpha-Kdo-(2->6)-lipid A + ADP + H+ (EC 2.4.99.23, RHEA:74067).
Justification: GO:0008920 has children for the heptosyltransferase II and III and lipid A-core reactions but none for the first heptose transfer performed by WaaC, which is why curators reach for ill-fitting siblings.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005829 cytosol | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: IBA from PTN001575314, seeded by E. coli WaaF (P37692), which is found in cytosolic proteome fractions. Reason: WaaC is a soluble enzyme that associates peripherally with the cytoplasmic face of the inner membrane, where its lipid acceptor is; it is cytoplasmic in the broad sense, but the activity is exercised at the membrane surface. Kept as a non-core location. Supporting Evidence: file:PSEAE/waaC/waaC-uniprot.txt SUBCELLULAR LOCATION: Cell inner membrane {ECO:0000250|UniProtKB:P24173}; Peripheral membrane protein {ECO:0000250|UniProtKB:P24173}; Cytoplasmic side {ECO:0000250|UniProtKB:P24173}. |
| GO:0008713 ADP-heptose-lipopolysaccharide heptosyltransferase activity | IBA GO_REF:0000033 | MODIFY | Summary: IBA from PTN001575314, seeded only by E. coli WaaF (P37692). GO:0008713 is defined as the heptosyltransferase II reaction (heptosyl-Kdo2-lipid A acceptor), which WaaF performs and WaaC does not. Reason: WaaC transfers the first heptose to Kdo2-lipid A, a different acceptor from the one in the GO:0008713 definition. GO has no term for the Hep I reaction, so the most specific correct term is the parent GO:0008920 lipopolysaccharide heptosyltransferase activity, which this gene already carries (InterPro IEA). The node is too broad for the Hep II term (GO_CENTRAL defect below). Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH WRONG ORTHOLOG OR PARALOG Sources checked: PANTHER:PTN001575314 · PTN001575314 SUPPORTS SOURCE BUT NOT TARGET Node spanning WaaC (Hep I), WaaF (Hep II) and WaaQ (Hep III) orthologs, as its own GO:0009244 seed list shows. The Hep II reaction term fits only the WaaF subclade. UniProtKB:P37692 · E. coli WaaF (IDA/EXP GO:0008713) SUPPORTS SOURCE BUT NOT TARGET Correct for WaaF itself; WaaC is a paralogous heptosyltransferase with a different acceptor. Proposed replacements: lipopolysaccharide heptosyltransferase activity Supporting Evidence: file:PSEAE/waaC/waaC-uniprot.txt Catalyzes the addition of the first heptose unit to one 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the Kdo2-lipid A module PMID:9171387 Two plasmids, pCOREc1 and pCOREc2, which restored smooth LPS production in the waaC mutant, were isolated. |
| GO:0009244 lipopolysaccharide core region biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: IBA from PTN001575314 (seven seeds: E. coli and Salmonella WaaC and WaaF, E. coli WaaQ, and PAO1 WaaC and WaaF). WaaC appearing among its own sources reflects its own PseudoCAP row. Reason: Core heptosyltransferases catalyse steps of inner-core assembly. The node term suits a clade of LPS core heptosyltransferases and the IBD stands on five enterobacterial IMP seeds. Supporting Evidence: PMID:9171387 Two plasmids, pCOREc1 and pCOREc2, which restored smooth LPS production in the waaC mutant, were isolated. |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Mapping of the UniProt location (cell inner membrane, peripheral, cytoplasmic side), itself by similarity to E. coli WaaC. Reason: WaaC acts on a lipid acceptor in the inner membrane and E. coli WaaC is membrane-associated, so the location holds by orthology; it is peripheral to the description of the enzyme's activity. Supporting Evidence: file:PSEAE/waaC/waaC-uniprot.txt SUBCELLULAR LOCATION: Cell inner membrane {ECO:0000250|UniProtKB:P24173}; Peripheral membrane protein {ECO:0000250|UniProtKB:P24173}; Cytoplasmic side {ECO:0000250|UniProtKB:P24173}. |
| GO:0008920 lipopolysaccharide heptosyltransferase activity | IEA GO_REF:0000002 | ACCEPT | Summary: InterPro2GO mapping of IPR011908 (LPS heptosyltransferase I family). Reason: The most specific correct GO term for heptosyltransferase I, since GO has no term for the Kdo2-lipid A acceptor reaction. Supported by family membership (NCBIfam TIGR02193 heptsyl_trn_I) and by complementation of the Salmonella waaC mutant. Supporting Evidence: file:PSEAE/waaC/waaC-uniprot.txt NCBIfam; TIGR02193; heptsyl_trn_I; 1. PMID:9171387 Two plasmids, pCOREc1 and pCOREc2, which restored smooth LPS production in the waaC mutant, were isolated. |
| GO:0009244 lipopolysaccharide core region biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: Electronic row for LPS core biosynthesis, consistent with the IBA and PseudoCAP rows. Reason: Same claim as the accepted IBA and experimental rows. Supporting Evidence: PMID:9171387 Two plasmids, pCOREc1 and pCOREc2, which restored smooth LPS production in the waaC mutant, were isolated. |
| GO:0016757 glycosyltransferase activity | IEA GO_REF:0000002 | TOO GENERAL | Summary: InterPro2GO mapping of the GT9 family signature. Reason: True, but entailed by the accepted GO:0008920 lipopolysaccharide heptosyltransferase activity, which is_a glycosyltransferase activity. |
| GO:0005886 plasma membrane | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: UniProt ISS transfer of the plasma membrane location from E. coli WaaC (P24173, EXP). Reason: Consistent with the IEA row: peripheral inner-membrane association by orthology to E. coli WaaC. Supporting Evidence: file:PSEAE/waaC/waaC-uniprot.txt SUBCELLULAR LOCATION: Cell inner membrane {ECO:0000250|UniProtKB:P24173}; Peripheral membrane protein {ECO:0000250|UniProtKB:P24173}; Cytoplasmic side {ECO:0000250|UniProtKB:P24173}. |
| GO:0009244 lipopolysaccharide core region biosynthetic process | IDA PMID:9171387 Isolation and characterization of two genes, waaC (rfaC) and... | ACCEPT | Summary: PseudoCAP row and a seed of the PTN001575314 IBD. The PAO1 gene restores smooth LPS to a Salmonella waaC mutant. Reason: Heterologous complementation of a heptosyltransferase I mutant places WaaC in inner-core assembly, and the enzyme catalyses a step of it. The evidence is genetic rather than a direct assay, so the IDA code is probably wrong (IGI or IMP); recorded as a suspected evidence-code defect because only the abstract was read. The term is sound. Supporting Evidence: PMID:9171387 Two plasmids, pCOREc1 and pCOREc2, which restored smooth LPS production in the waaC mutant, were isolated. |
| GO:0071968 lipid A-core heptosyltransferase activity | ISS PMID:9171387 Isolation and characterization of two genes, waaC (rfaC) and... | MODIFY | Summary: PseudoCAP ISS from Salmonella WaaC (P26469). GO:0071968 is the reaction that completes lipid A-core (galactosyl-glucosyl3-heptosyl3-Kdo2-lipid A acceptor), not the first heptose transfer. Reason: The with/from protein is heptosyltransferase I, which adds the first heptose to Kdo2-lipid A; the asserted term names a late outer-core heptose transfer that neither WaaC performs. The sound part of the claim (an LPS heptosyltransferase) is GO:0008920, already accepted for this gene. Proposed replacements: lipopolysaccharide heptosyltransferase activity Supporting Evidence: file:PSEAE/waaC/waaC-uniprot.txt Catalyzes the addition of the first heptose unit to one 3-deoxy-D-manno-octulosonic acid (Kdo) residue of the Kdo2-lipid A module |
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Experiment: Conditional depletion of waaC in PAO1 with analysis of growth, LPS core structure by MS, and outer membrane permeability; in vitro Hep I assay with purified WaaC, ADP-heptose and Kdo2-lipid A.
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