DadX is an alanine-specific, pyridoxal-phosphate-dependent racemase that reversibly interconverts L-alanine and D-alanine. It is encoded beside dadA in a conserved catabolic locus, where D-alanine is proposed to be oxidized to pyruvate. DadX is the leading candidate for cytoplasmic D-alanine supply in KT2440, but a direct contribution to peptidoglycan synthesis has not been demonstrated.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0003824 catalytic activity | IEA GO_REF:0000002 | MARK AS OVER ANNOTATED | Summary: The generic catalytic term is superseded by alanine racemase activity. Reason: GO:0008784 captures the experimentally established reaction. Supporting Evidence: PMID:23995642 Conversely, the putative catabolic alanine racemase DadX showed narrow substrate specificity, clearly preferring only the alanine stereoisomers as the substrates. |
| GO:0005829 cytosol | IEA GO_REF:0000118 | ACCEPT | Summary: Cytosolic localization is plausible and consistent with the protein record. Reason: TreeGrafter supplies the annotation, and the mature protein record lacks the signal peptide that distinguishes the periplasmic KT2440 Alr/BSR. No direct fractionation experiment for DadX was identified. |
| GO:0008784 alanine racemase activity | IEA GO_REF:0000120 | ACCEPT | Summary: This is the exact, experimentally corroborated activity. Reason: DadX reversibly and selectively racemizes alanine stereoisomers. Supporting Evidence: PMID:23995642 Conversely, the putative catabolic alanine racemase DadX showed narrow substrate specificity, clearly preferring only the alanine stereoisomers as the substrates. |
| GO:0030170 pyridoxal phosphate binding | IEA GO_REF:0000120 | KEEP AS NON CORE | Summary: PLP is a supported catalytic cofactor rather than the core function. Reason: UniProt records the covalent PLP-linked lysine at residue 33. Supporting Evidence: file:PSEPK/dadX/dadX-uniprot.txt N6-(pyridoxal phosphate)lysine |
| GO:0030632 D-alanine biosynthetic process | IEA GO_REF:0000120 | UNDECIDED | Summary: DadX forms D-alanine, but its physiological process assignment is unresolved. Reason: The reversible chemistry can produce D-alanine, whereas the characterized locus and literature frame DadX as catabolic. No dadX genetic, flux, or peptidoglycan experiment establishes cell-wall D-alanine supply, so this process annotation is not promoted into the core function. Supporting Evidence: PMID:23995642 P. putida KT2440 catabolized the d-stereoisomers of lysine, phenylalanine, arginine, alanine, and hydroxyproline as the sole carbon and nitrogen sources. PMID:23995642 However, DadX did show 6- and 9-fold higher k(cat)/K(m) values than Alr with l- and d-alanine, respectively. |
| GO:0008784 alanine racemase activity | EXP PMID:23995642 Amino acid racemization in Pseudomonas putida KT2440. | ACCEPT | Summary: Direct biochemical characterization establishes alanine racemase activity. Reason: The purified enzyme selectively catalyzed both directions of alanine racemization. Supporting Evidence: PMID:23995642 Conversely, the putative catabolic alanine racemase DadX showed narrow substrate specificity, clearly preferring only the alanine stereoisomers as the substrates. |
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Download this section (compressed HTML)Q: Does DadX provide the cytoplasmic D-alanine used for peptidoglycan synthesis in KT2440?
Experiment: Deplete or delete dadX and measure growth, peptidoglycan composition, intracellular D-alanine, and rescue by exogenous D-alanine; include isotope tracing from L-alanine into the terminal D-Ala-D-Ala moiety.
Type: genetics, metabolite rescue, peptidoglycan analysis, and isotope tracing
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