WbpY (PA5448) is a 375-residue GT4-family (GT-B fold) D-rhamnosyltransferase of Pseudomonas aeruginosa PAO1 encoded in the common polysaccharide antigen (CPA, A-band) gene cluster. CPA is a D-rhamnan built from [D-Rha a1-2 D-Rha a1-3 D-Rha a1-3] trisaccharide repeats on an undecaprenyl-diphosphate-linked GlcNAc primer made by WbpL and capped with the first a1-3-linked D-Rha by WbpZ. WbpY transfers D-rhamnose from GDP-D-rhamnose in a1-3 linkage onto the WbpZ product, and together with WbpX (which adds a1-2- and a1-3-linked D-Rha) extends the D-rhamnan in a non-processive manner. wbpY mutants lack A-band LPS but still make B-band (O-specific antigen) LPS.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0009103 lipopolysaccharide biosynthetic process | IBA GO_REF:0000033 | MODIFY | Summary: IBA lipopolysaccharide biosynthetic process from PTN000749272, whose sole seed is the PseudoCAP IMP row on WbpY itself. Reason: The IBD is sound at the node (WbpY's own appearance in WITH/FROM reflects the experimental seed and is not circular), and LPS biosynthesis is an appropriate breadth for a 19-taxon GT4 subclade. For WbpY itself the precise process is known; it extends the CPA O-antigen chain, and GO:0009243 O antigen biosynthetic process (part_of LPS biosynthetic process) is already carried by this gene (IDA). The row is refined accordingly; no change is requested at the node. Propagation Review Root cause: NO FAILURE CORE Sources checked: UniProtKB:Q9HTB9 SUPPORTS TRANSFER Sole seed is the PseudoCAP IMP row (PMID:9680202; wbpY mutants lack A-band LPS). Later biochemistry (PMID:35166992) shows WbpY catalyses a CPA chain-extension step. Proposed replacements: O antigen biosynthetic process Supporting Evidence: PMID:9680202 these mutants are unable to synthesize A-band LPS, while B-band synthesis is unaffected PMID:35166992 NMR spectroscopic analysis of the WbpY reaction product showed that WbpY transferred one D-Rha residue in Ξ±1-3 linkage |
| GO:0016757 glycosyltransferase activity | IBA GO_REF:0000033 | MODIFY | Summary: IBA glycosyltransferase activity from PTN005302544, seeded by PseudoCAP IMP rows on WbpX (G3XCU3) and WbpY. Reason: Glycosyltransferase activity is correct for WbpY and an apt breadth for the 60-leaf GT4 node, so the IBD conclusion stands even though both seeds are IMP rows from the 1998 genetic paper. WbpY and WbpX were later shown directly to transfer D-Rha from GDP-D-Rha (PMID:35166992), so the node should be re-seeded with that IDA. For WbpY itself the activity is a D-rhamnosyltransferase; GO has no D-rhamnosyltransferase term and places rhamnosyltransferases under GO:0016758 hexosyltransferase activity, the most specific existing term. Propagation Review Root cause: NO FAILURE CORE Failure modes: SOURCE EVIDENCE WEAK Sources checked: UniProtKB:Q9HTB9 SOURCE WEAK OR INFERRED Seed is a PseudoCAP IMP MF row from a mutant LPS phenotype (PMID:9680202); the activity is true (PMID:35166992), only the seed provenance is weak. UniProtKB:G3XCU3 SOURCE WEAK OR INFERRED WbpX co-seed from the same 1998 genetic paper; WbpX D-RhaT activity was shown in PMID:35166992. Proposed replacements: hexosyltransferase activity Supporting Evidence: PMID:35166992 We demonstrated that these GTs are indeed D-RhaTs and catalyze the transfer of D-Rha from GDP-D-Rha to the reaction product of WbpZ. |
| GO:0016757 glycosyltransferase activity | IEA GO_REF:0000120 | MODIFY | Summary: Combined IEA from the glycosyl transferase family 1 domain (IPR001296) and ARBA. Reason: Correct (GT4 domains PF13439/PF00534), but WbpY is demonstrated to be a D-rhamnosyltransferase; refined to GO:0016758 hexosyltransferase activity as for the other glycosyltransferase rows. Proposed replacements: hexosyltransferase activity Supporting Evidence: file:PSEAE/wbpY/wbpY-uniprot.txt Glycosyl transferase family 1 PMID:35166992 WbpX and WbpY recombinantly expressed in E. coli differ in their donor and acceptor specificities and have properties of GT-B folded enzymes of the GT4 glycosyltransferase family |
| GO:0009103 lipopolysaccharide biosynthetic process | IMP PMID:9680202 Three rhamnosyltransferases responsible for assembly of the ... | MODIFY | Summary: PseudoCAP IMP LPS biosynthetic process (wbpY mutant lacks A-band LPS); sole seed of PTN000749272. Reason: The seed is sound in substance; the mutant lacks A-band LPS with B-band unaffected and WbpY catalyses a step of CPA synthesis. The specific process is CPA O-antigen synthesis, GO:0009243 O antigen biosynthetic process (part_of LPS biosynthetic process), which this gene already carries. Proposed replacements: O antigen biosynthetic process Supporting Evidence: PMID:9680202 Chromosomal wbpX, wbpY and wbpZ mutants were generated, and Western immunoblot analysis demonstrates that these mutants are unable to synthesize A-band LPS, while B-band synthesis is unaffected. |
| GO:0009243 O antigen biosynthetic process | IDA PMID:9680202 Three rhamnosyltransferases responsible for assembly of the ... | ACCEPT | Summary: PseudoCAP IDA O antigen biosynthetic process from the 1998 A-band cluster paper. Reason: Core process. The abstract describes mutant immunoblots (an IMP-type experiment); the full text is not available, so the IDA code is not challenged here. The claim is independently confirmed by the in vitro D-rhamnosyltransferase activity of WbpY on a CPA primer (PMID:35166992). Supporting Evidence: PMID:9680202 The A-band polysaccharide is assembled by the alpha-D-rhamnosyltransferases, WbpX, WbpY and WbpZ. PMID:35166992 the combination of WbpX and WbpY resulted in the synthesis of a D-Rhamnan polymer of at least 8 D-Rha residues |
| GO:0016757 glycosyltransferase activity | IMP PMID:9680202 Three rhamnosyltransferases responsible for assembly of the ... | MODIFY | Summary: PseudoCAP IMP glycosyltransferase activity from the wbpY mutant phenotype; seed of PTN005302544. Reason: The activity is real (direct D-RhaT assays, PMID:35166992) but the 1998 evidence is a mutant LPS phenotype combined with sequence similarity (per the IMP code and the abstract; full text not read), which does not show the molecular activity; the abstract itself describes the transferases as predicted. Refined to the specific supported term GO:0016758 hexosyltransferase activity; PseudoCAP is asked to re-key the evidence to the direct assay. Proposed replacements: hexosyltransferase activity Supporting Evidence: PMID:9680202 The last two transferases are predicted to be processive PMID:35166992 We demonstrated that these GTs are indeed D-RhaTs and catalyze the transfer of D-Rha from GDP-D-Rha to the reaction product of WbpZ. |
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Download this section (compressed HTML)Q: Should GO add a D-rhamnosyltransferase activity term (GDP-D-rhamnose donor, distinct from the plant/bacterial L-rhamnosyltransferases that use UDP- or dTDP-L-rhamnose)?
Q: Does WbpY act at the cytoplasmic face of the inner membrane in a complex with WbpX, WbpZ and the Wzm/Wzt ABC transporter, as for other ABC-transporter-dependent O antigens?
Experiment: Determine the in vivo order of WbpY and WbpX additions by analysing CPA chains from wbpY and wbpX point mutants (catalytic EX7E motif) complemented in trans, and test whether a WbpX-WbpY complex is required for full-length chain synthesis.
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