PA1268 (LhpA) is the 4-hydroxyproline 2-epimerase of Pseudomonas aeruginosa, a PLP-independent member of the proline racemase family. It interconverts trans-4-hydroxy-L-proline and cis-4-hydroxy-D-proline using a pair of active-site cysteines (Cys88 and Cys236), one from each subunit of the homodimer, and has no detectable proline racemase activity. The reaction is the entry step of bacterial hydroxyproline catabolism, which delivers cis-4-hydroxy-D-proline to D-hydroxyproline dehydrogenase and onward to 2-oxoglutarate. lhpA lies in a hydroxyproline-inducible locus controlled by the AraC-family regulator LhpR, and the recombinant protein is also reported to act as a B-cell mitogen.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0047580 4-hydroxyproline epimerase activity | EXP PMID:17849014 Molecular and structural discrimination of proline racemase ... | ACCEPT | Summary: Purified PaHyPRE epimerizes 4-hydroxyproline and lacks proline racemase activity; this is the core activity. Reason: The recombinant P. aeruginosa protein was assayed with both proline and 4-hydroxyproline stereoisomers. It epimerized 4-hydroxy-L/D-proline, had no proline racemase activity, and lost activity when either catalytic cysteine was mutated, which ties the activity to the protein itself. Supporting Evidence: PMID:17849014 B. pseudomallei and P. aeruginosa recombinant proteins presented no measurable PRAC activity but demonstrated strong epimerization of OH-L/D-Pro behaving as genuine OH-Pro epimerases. PMID:17849014 In comparison to wild type HyPRE, C88SHyPRE and C236SHyPRE single mutations induce radical loss of OH-L/D-Pro epimerization establishing that proton transfer during HyPRE reaction is indeed dependent on the presence of the catalytic βCys-Cysβ couple of each subunit (Figure 6). |
| GO:0047580 4-hydroxyproline epimerase activity | EXP PMID:20665524 Determination of 4-hydroxyproline-2-epimerase activity by ca... | ACCEPT | Summary: A capillary-electrophoresis assay measured 4-hydroxyproline 2-epimerase kinetics; consistent with the core activity. Reason: Only the abstract is cached, and it does not name the source organism of the model enzyme. UniProt curators, who read the full text, attribute the trans-L and cis-D Km values to this protein. The activity agrees with the direct assays in PMID:17849014, so the annotation is accepted and the attribution is left to the curator. Supporting Evidence: PMID:20665524 Enzyme activity and competition assays were evaluated for various hydroxyproline diastereoisomers, proline enantiomers and their structural analogs using 4-hydroxyproline-2-epimerase as a model system. file:PSEAE/PA1268/PA1268-uniprot.txt KM=2.4 mM for trans-4-hydroxy-L-proline {ECO:0000269|PubMed:20665524}; |
| GO:0047580 4-hydroxyproline epimerase activity | EXP PMID:24980702 Prediction and characterization of enzymatic activities guid... | ACCEPT | Summary: The proline racemase superfamily survey places this protein's cluster among the characterized 4-hydroxyproline epimerases. Reason: The study lists Q9I476 among the proteins it functionally characterized and assigns the 4HypE function to the cluster containing the 2AZP structure of this protein. The same study shows that P. aeruginosa PAO1 grows on trans-4-hydroxy-L-proline. Supporting Evidence: PMID:24980702 As a second retrospective example, the 4HypE function has been assigned to members of the blue (cluster 1) and red (cluster 2) clusters in the SSN (Figure 2B). PMID:24980702 These included, 2AZP, a putative 4HypE (from cluster 2) determined unliganded by the Midwest Center for Structural Genomics |
| GO:0047580 4-hydroxyproline epimerase activity | IEA GO_REF:0000120 | ACCEPT | Summary: The automated EC/Rhea mapping repeats the experimentally established epimerase activity. Reason: The electronic row restates the same reaction (Rhea 21152, EC 5.1.1.8) that the experimental rows establish. It is concordant with them and is accepted on the strength of the experimental evidence, not the ARBA rule. Supporting Evidence: file:PSEAE/PA1268/PA1268-uniprot.txt Reaction=trans-4-hydroxy-L-proline = cis-4-hydroxy-D-proline; Xref=Rhea:RHEA:21152, ChEBI:CHEBI:57690, ChEBI:CHEBI:58375; EC=5.1.1.8; Evidence={ECO:0000269|PubMed:17849014, |
| GO:0047580 4-hydroxyproline epimerase activity | IMP PMID:27145750 Molecular characterization of LhpR in control of hydroxyprol... | ACCEPT | Summary: Genetics of the lhp loci identify LhpA as one of two hydroxyproline epimerases needed for D-Hyp signalling. Reason: Only the abstract is cached. It names LhpA as the Hyp epimerase and shows that induction of the lhpA promoter by D-hydroxyproline is lost in the lhpA lhpK double mutant, consistent with LhpA converting D-Hyp to the L-Hyp signal. This mutant phenotype corroborates the biochemically established activity, so the row is accepted. Supporting Evidence: PMID:27145750 The first locus includes lhpABFE encoding a Hyp epimerase (LhpA) and d-Hyp dehydrogenase (LhpBEF) 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. |
| GO:0019470 trans-4-hydroxy-L-proline catabolic process | IMP PMID:27145750 Molecular characterization of LhpR in control of hydroxyprol... | NEW | Summary: LhpA catalyses the first step of hydroxyproline catabolism, and lhp knockouts impair growth on L-Hyp. Reason: The GOA rows give only the molecular function. LhpA performs the first chemical step of the route, epimerizing trans-4-hydroxy-L-proline to the cis-D substrate of D-hydroxyproline dehydrogenase. That is participation in the process, not just being required for it. Knockout growth phenotypes in the lhp loci, and P. aeruginosa growth on trans-4-hydroxy-L-proline, place that step in L-Hyp catabolism. KT2440 ProR carries the same process term in its review. Supporting Evidence: PMID:27145750 Utilization of hydroxy-l-proline (l-Hyp) in Pseudomonas aeruginosa requires conversion of l-Hyp to d-Hyp followed by the d-Hyp dehydrogenase pathway PMID:27145750 The importance of these genes on l-Hyp catabolism was supported by growth phenotype analysis on knockout mutants. PMID:22833679 Pseudomonas putida and Pseudomonas aeruginosa convert L-hydroxyproline to Ξ±-ketoglutarate via four hypothetical enzymatic steps different from known mammalian pathways |
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Download this section (compressed HTML)Q: Is the reported B-cell mitogenic activity of recombinant P. aeruginosa HyPRE a property of the native enzyme during infection, or an effect of the purified recombinant protein?
Q: Which PAO1 genes encode the three subunits of the LhpBEF D-hydroxyproline dehydrogenase that acts on this enzyme's product? PMID:27145750 gives the operon as lhpABFE and lhpA is PA1268, so the subunits should be among the adjacent genes; a UniProt query returns no gene named lhpB, lhpE or lhpF in PAO1, leaving the adjacent loci PA1267 and PA1266, whose entries carry only uninformative protein names, as candidates with no third subunit identified.
Experiment: Compare growth on trans-4-hydroxy-L-proline and cis-4-hydroxy-D-proline in single lhpA and lhpK mutants and the double mutant, with stereoisomer-resolved metabolite measurements, to define how much epimerase flux each paralog carries.
Type: paralog-resolved genetics and metabolite profiling
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