MphA (macrolide 2'-phosphotransferase I, MPH(2')I) is a macrolide kinase that inactivates macrolide antibiotics by transferring the gamma-phosphate of a purine nucleoside triphosphate (GTP, ITP or ATP, with GTP favored) to the 2'-hydroxyl of the desosamine sugar, producing an inactive macrolide 2'-O-phosphate that no longer binds the bacterial ribosome. In contrast to the broad-spectrum MphB (MPH(2')II), MphA is comparatively narrow: it acts efficiently on 14-membered (erythromycin, oleandomycin, clarithromycin, roxithromycin) and 15-membered (azithromycin) macrolides but only very weakly on 16-membered macrolides (spiramycin, josamycin, tylosin). A second distinction is regulation: MphA synthesis is inducible by erythromycin via the upstream TetR-family repressor MphR(A), whereas MphB is constitutive. High-level erythromycin resistance from the original determinant requires mphA together with the adjacent mrx gene (an accessory membrane protein). The enzyme adopts the bi-lobed protein-kinase-like fold of the aminoglycoside phosphotransferase (APH) superfamily (crystal structures solved for MPH(2')-I with a guanine nucleotide and macrolides). MphA is the most clinically prevalent plasmid-borne macrolide-resistance determinant in Enterobacterales and is frequently associated with reduced susceptibility to azithromycin. In contemporary isolates it is typically carried as a mobile IS26/IS6100-bounded composite transposon spanning the mphA-mrx(A)-mphR(A) operon, predominantly on IncF plasmids, which drives its wide horizontal dissemination.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0050073 macrolide 2'-kinase activity | IDA PMID:2478074 Purification and characterization of macrolide 2'-phosphotra... | NEW | Summary: MphA phosphorylates the 2'-OH of macrolides using a purine NTP, producing inactive macrolide 2'-O-phosphate. The purified enzyme is highly active on 14-membered (and 15-membered) macrolides; this matches the GO:0050073 definition (ATP + oleandomycin = ADP + 2 H+ + oleandomycin 2'-O-phosphate). Reason: The purified enzyme was biochemically characterized as a macrolide 2'-phosphotransferase, and crystal structures of MPH(2')-I confirm the kinase mechanism. No curated GOA annotation exists for this TrEMBL entry, so this specific MF should be added. Supporting Evidence: PMID:2478074 MPH(2') is an inducible intracellular enzyme which showed high levels of activity with 14-member-ring macrolides and extremely low levels with 16-member-ring macrolides. PMID:28416110 We present structures for MPH(2')-I and MPH(2')-II in the apo state, and in complex with GTP analogs and six different macrolides. |
| GO:0046677 response to antibiotic | IMP PMID:8619599 Nucleotide sequence and characterization of erythromycin res... | NEW | Summary: MphA confers macrolide (notably erythromycin and azithromycin) resistance by enzymatically inactivating the drug; high-level resistance from the native determinant additionally requires mrx. Expression is inducible by erythromycin via the MphR(A) repressor. Reason: Resistance is the biological process this enzyme participates in. "response to antibiotic" is the standard, well-supported BP for an antibiotic-modifying resistance enzyme (the drug is modified, not degraded, so "antibiotic catabolic process" would be less accurate). Supporting Evidence: PMID:8619599 the expression of high-level resistance to erythromycin requires two genes, mphA and mrx, which encode macrolide 2'-phosphotransferase I and an unidentified hydrophobic protein, respectively. PMID:17302923 The mph(A) gene was unique in conferring resistance to azithromycin. PMID:38521802 The MIC of azithromycin was ≥ 256 µg/ml for all transconjugants. |
| GO:0005737 cytoplasm | IDA PMID:2478074 Purification and characterization of macrolide 2'-phosphotra... | NEW | Summary: MphA was characterized as a soluble, intracellular (cytoplasmic) enzyme upon purification from E. coli, consistent with cytoplasmic inactivation of macrolides. Reason: The purified enzyme was reported as an inducible intracellular enzyme, indicating cytoplasmic localization — the expected compartment for a soluble macrolide-inactivating kinase. Supporting Evidence: PMID:2478074 MPH(2') is an inducible intracellular enzyme |
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Download this section (compressed HTML)Q: What is the structural basis for MphA's discrimination against 16-membered macrolides, and could 16-membered macrolides or derivatives evade MphA-mediated resistance clinically?
Experiment: Measure steady-state kinetics (kcat/Km) of purified MphA against 14-, 15- and 16-membered macrolides with GTP vs ATP donors to quantify the substrate and cofactor preferences relative to MphB.
Experiment: Test whether the accessory mrx gene is required for full resistance in a clean E. coli background and determine its molecular role (e.g. membrane association / enzyme stability).
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