mphB

MphB (macrolide 2'-phosphotransferase II, MPH(2')II) is a plasmid-encoded macrolide kinase that inactivates macrolide antibiotics. It transfers the gamma-phosphate of a purine nucleoside triphosphate (ATP, GTP or ITP) to the 2'-hydroxyl group of the desosamine sugar of macrolides, producing an inactive macrolide 2'-O-phosphate that can no longer bind the bacterial ribosome; this enzymatic detoxification confers macrolide resistance. MphB acts on a broad range of substrates spanning 14-membered (e.g. oleandomycin, erythromycin, clarithromycin, roxithromycin, dirithromycin), 15-membered (azithromycin), and 16-membered (e.g. spiramycin, tylosin, josamycin) macrolides as well as the ketolide telithromycin (CARD ARO:3000318). Early phenotyping placed MphB as relatively narrow (with azithromycin resistance attributed specifically to MphA), but later enzymatic and mass-spectrometric work showed MphB inactivates essentially all macrolides tested, including azithromycin and telithromycin. The cloned mphB encodes a 302-residue, ~34.5 kDa protein matching this UniProt entry exactly. Crystal structures of the MphB class (MPH(2')-II) reveal the bi-lobed protein-kinase-like fold of the aminoglycoside phosphotransferase (APH) superfamily, distinguished by a large interdomain linker that forms an expanded, largely hydrophobic macrolide-binding pocket; nucleotide is bound in the kinase pocket (structures captured with GTP analogs, and GTP is a preferred donor in vitro, though ATP, GTP and ITP can all serve). Catalysis depends on conserved active-site residues — aspartates D200/D209/D219/ D231 and the conserved His205 — and on a divalent metal (activity is EDTA-inhibited), consistent with the Mg2+-dependent phosphotransfer chemistry of this fold; D227 contributes to recognition of 16-membered macrolides. In this organism the gene is carried on plasmid pO83_CORR of the adherent-invasive Escherichia coli strain NRG 857C, making it part of the mobile macrolide-resistance gene pool of enteric bacteria.