lhpL (PA1255) encodes a 344-residue bifunctional enzyme of the proline racemase family (Pfam PF05544; catalytic cysteine pair) that acts on trans-3-hydroxy-L-proline. It dehydrates trans-3-hydroxy-L-proline to Delta(1)-pyrroline-2-carboxylate (EC 4.2.1.77) and also 2-epimerizes it to cis-3-hydroxy-D-proline. It has no activity on L-proline, on the 4-hydroxyprolines, or on D-proline, so it is not a proline racemase and not a 4-hydroxyproline 2-epimerase; the latter activity in this organism belongs to a separate gene, PA1268. lhpL sits in the hydroxyproline (lhp) gene cluster of Pseudomonas aeruginosa PAO1, is induced by trans-4- and trans-3-hydroxy-L-proline as sole carbon source under the control of the AraC-family activator LhpR, and is thought to let the organism funnel trans-3-hydroxy-L-proline, which is released from hydroxyproline-containing peptide antibiotics and from collagen-derived material, into proline and so into central metabolism.
Definition: Catalysis of the reaction: trans-3-hydroxy-L-proline = cis-3-hydroxy-D-proline.
Justification: Swiss-Prot records this reaction for Q9I489 with experimental evidence and an incomplete EC number (5.1.1.-), but no GO term exists for it, so half of the gene's curated bifunctional activity cannot be annotated. It is distinct from GO:0047580 (the 4-hydroxy isomer) and from GO:0018112 proline racemase activity.
Parent term: racemase and epimerase activity, acting on amino acids and derivatives
Supporting Evidence:
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0047580 4-hydroxyproline epimerase activity | IBA GO_REF:0000033 | MODIFY | Summary: IBA from PTN002433936, an internal duplication node of PTHR33442 with 9 leaves in 7 organisms, sole-seeded by the PseudoCAP IMP row on this gene. The clade is coherent (proline racemase family; one Bacillus cereus leaf and the Nematostella leaf are themselves named trans-3-hydroxy-L-proline dehydratases), but the term is the 4-hydroxy reaction, which has been assayed on this protein and is absent. Reason: The essence is right (an epimerase/dehydratase acting on a hydroxyproline) but the isomer is wrong. GO:0047580 is trans-4-hydroxy-L-proline = cis-4-hydroxy-D-proline; UniProt records experimentally that this protein displays no t4LHyp epimerase activity, and its characterized reactions are on trans-3-hydroxy-L-proline. GO:0050346, which the gene already carries by EXP, is the supported term for the dehydratase half; the 2-epimerase half has no GO term yet (proposed below). Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: FUNCTIONAL DIVERGENCE Sources checked: PANTHER:PTN002433936 · PTN002433936 SOURCE BAD The node is a sound proline-racemase-family clade, but its IBD term should be GO:0050346 rather than GO:0047580. GOA holds 35 GO:0047580 rows with this node in with/from (9 GO_Central, 24 TreeGrafter, 2 UniProt). UniProtKB:Q9I489 · lhpL (PA1255) SOURCE BAD Sole seed. The seed is the PseudoCAP IMP row, an induction phenotype of an lhpA lhpK double mutant, and the 4-hydroxy reaction is experimentally absent on this protein. Proposed replacements: trans-L-3-hydroxyproline dehydratase activity Supporting Evidence: file:PSEAE/lhpL/lhpL-uniprot.txt Displays neither t4LHyp epimerase nor proline racemase activity (PubMed:17849014). file:PSEAE/lhpL/lhpL-bioinformatics/RESULTS.md PTN002433936 is an internal DUPLICATION node of PTHR33442. |
| GO:0047580 4-hydroxyproline epimerase activity | IEA GO_REF:0000117 | MODIFY | Summary: ARBA association rule for the same term. The rule was almost certainly learnt from proline-racemase-family entries annotated with the 4-hydroxyproline reaction, so it repeats the mis-assignment rather than providing independent support. Reason: Same judgment as the IBA and IMP rows for this term: the protein acts on the 3-hydroxy isomer, and the 4-hydroxy epimerization is experimentally absent, so the term should be GO:0050346. Proposed replacements: trans-L-3-hydroxyproline dehydratase activity Supporting Evidence: file:PSEAE/lhpL/lhpL-uniprot.txt Reaction=trans-3-hydroxy-L-proline = 1-pyrroline-2-carboxylate + H2O; |
| GO:0047580 4-hydroxyproline epimerase activity | IMP PMID:27145750 Molecular characterization of LhpR in control of hydroxyprol... | MODIFY | Summary: PseudoCAP IMP, the sole seed of the PTN002433936 IBD. Li & Lu call the gene at PA1255 lhpK and describe it from sequence as a "Hyp/Pro racemase"; their genetic result is that induction of the lhpA promoter by D-Hyp is lost in an lhpA lhpK double mutant. That is an induction phenotype of two genes together, and as the abstract describes it, it cannot say which reaction either protein catalyses or which hydroxyproline isomer is involved (the full text was not read). Reason: The gene is correctly placed in hydroxyproline catabolism, but the term names a reaction (trans-4-hydroxy-L-proline = cis-4-hydroxy-D-proline) that was assayed on this protein and not found. The curated activity is on trans-3-hydroxy-L-proline, and PAO1's 4-hydroxyproline 2-epimerase is PA1268, which carries its own GO:0047580 IMP from this same paper. Proposed replacements: trans-L-3-hydroxyproline dehydratase activity Supporting Evidence: PMID:27145750 the second locus codes for a putative ABC transporter (LhpPMNO), a protein of unknown function (LhpH), Hyp/Pro racemase (LhpK) and two enzymes in l-Hyp catabolism (LhpC and LhpG) PMID:27145750 Induction of the lhpA promoter by d-Hyp was completely abolished in the lhpA lhpK double mutant devoid of two epimerases, while the induction effect of l-Hyp remained unchanged file:PSEAE/lhpL/lhpL-uniprot.txt Displays neither t4LHyp epimerase nor proline racemase activity (PubMed:17849014). |
| GO:0050346 trans-L-3-hydroxyproline dehydratase activity | EXP PMID:27929065 Functional characterization of aconitase X as a cis-3-hydrox... | ACCEPT | Summary: Direct experimental assignment of the dehydration of trans-3-hydroxy-L-proline to Delta(1)-pyrroline-2-carboxylate, with kinetics (KM 23 mM, kcat 28.7 min-1) curated by Swiss-Prot. This is the gene's characterized activity. Reason: Core molecular function, directly demonstrated for this protein. Supporting Evidence: PMID:27929065 We herein demonstrated that PaLhpL gene (PA1255; Fig. 1d) in the L-hydroxyproline gene cluster encodes a bifunctional dehydratase and 2-epimerase toward trans-3-hydroxy-L-proline as the leading candidate file:PSEAE/lhpL/lhpL-uniprot.txt Reaction=trans-3-hydroxy-L-proline = 1-pyrroline-2-carboxylate + H2O; |
| GO:0050346 trans-L-3-hydroxyproline dehydratase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Automatic mapping from the curated EC number and Rhea reaction on the UniProt entry. It agrees with the EXP row. Reason: Correct and as specific as the evidence allows; reinforces the experimental row for the same term. Supporting Evidence: file:PSEAE/lhpL/lhpL-uniprot.txt Reaction=trans-3-hydroxy-L-proline = 1-pyrroline-2-carboxylate + H2O; |
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Download this section (compressed HTML)Q: Should PTN002433936's IBD be re-pointed from GO:0047580 to GO:0050346, and should the 35 propagated rows (including TreeGrafter transfers) be re-derived from the corrected node?
Q: Is there a GO term, or should one be created, for the catabolic pathway that converts trans-3-hydroxy-L-proline to L-proline, as a sibling of GO:0019470 trans-4-hydroxy-L-proline catabolic process?
Experiment: Delete PA1255 alone in PAO1 and test growth on trans-3-hydroxy-L-proline, cis-3-hydroxy-L-proline and trans-4-hydroxy-L-proline as sole carbon sources, with the PA1268 deletion as comparator. This would separate the two epimerase/dehydratase genes physiologically, which the published double-mutant induction assay cannot do.
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