Cloning and nucleotide sequence of the mphB gene for macrolide 2'-phosphotransferase II in Escherichia coli
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The mphB gene encoding MPH(2')II was cloned and sequenced from E. coli; it encodes a 302-aa protein of 34483 Da, matching this UniProt entry (302 aa, ~34485 Da).
"mphB encoded a protein of 302 amino acids with a molecular mass of 34483 Da."
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The C-terminal region resembles the conserved functional domain of aminoglycoside phosphotransferases (the APH/protein-kinase-like fold).
"The carboxy terminal region of the deduced protein contained a sequence that resembled a conserved functional domain in aminoglycoside phosphotransferases."
Resistance phenotypes conferred by macrolide phosphotransferases
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mph genes were assayed in a macrolide/ketolide-susceptible E. coli AG100A background (AcrAB efflux pump disrupted), enabling clean comparison of phosphotransferase-conferred resistance.
"The mph genes were cloned into Escherichia coli AG100A susceptible to macrolides and ketolides following disruption of the AcrAB pump."
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mph(B) confers resistance to spiramycin (16-membered) and, like the other mph genes, to telithromycin.
"The mph(C) gene, as reported for mph(B), also conferred resistance to spiramycin...The four investigated genes conferred resistance to telithromycin."
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In this 2007 comparison mph(A) was reported as uniquely conferring azithromycin resistance — the older narrow-spectrum view of mph(B) later revised by Pawlowski et al. 2018 (PMID:29317655).
"The mph(A) gene was unique in conferring resistance to azithromycin."
Purification and characterization of macrolide 2'-phosphotransferase type II from a strain of Escherichia coli highly resistant to macrolide antibiotics
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MPH(2')II inactivates macrolides by phosphorylation of the 2'-hydroxyl; the inactivated oleandomycin product was identified as oleandomycin 2'-phosphate.
"Inactivated oleandomycin was identified as oleandomycin 2'-phosphate by thin-layer chromatography."
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MPH(2')II is a constitutive intracellular enzyme active on both 14- and 16-membered macrolides.
"MPH(2')II was a constitutive intracellular enzyme which showed high levels of activity with both 14-member-ring and 16-member-ring macrolides."
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Purine nucleotides (ITP, GTP, ATP) serve as phosphate donors; activity is metal-dependent (EDTA-inhibited).
"Purine nucleotides, such as ITP, GTP and ATP, were effective as cofactors in the inactivation of macrolides. An inhibitory effect of iodine, EDTA, or divalent cations on MPH(2')II activity was observed."
Expression of the mphB gene for macrolide 2'-phosphotransferase II from Escherichia coli in Staphylococcus aureus
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mphA and mphB encode macrolide 2'-phosphotransferases I and II and confer macrolide resistance in E. coli.
"The genes mphA and mphB encode macrolide 2'-phosphotransferases I and II, respectively, and they confer resistance to macrolide antibiotics in Escherichia coli."
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mphB conferred high-level resistance to the 16-membered-ring macrolide spiramycin when expressed in S. aureus.
"The gene endowed S. aureus with high-level resistance to spiramycin, a macrolide antibiotic with a 16-membered ring."
Identification of functional amino acids in the macrolide 2'-phosphotransferase II
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MPH(2') transfers the gamma-phosphate of ATP to the 2'-OH group of macrolide antibiotics.
"Macrolide 2'-phosphotransferase [MPH(2')] transfers the gamma phosphate of ATP to the 2'-OH group of macrolide antibiotics."
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Active-site aspartates D200, D209, D219 and D231 are essential for catalysis; D227 is non-essential and contributes to 16-membered-ring macrolide recognition.
"D200A, D209A, D219A, and D231A mutant strains were unable to inactivate the substrate oleandomycin, while a D227A mutant retained 7% of the activity of the original enzyme."
The role of histidine residues conserved in the putative ATP-binding region of macrolide 2'-phosphotransferase II
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MPH(2')II catalyzes transfer of the gamma-phosphate of ATP to the 2'-hydroxyl group of macrolides.
"Macrolide 2'-phosphotransferase (MPH(2')) catalyzes the transfer of the gamma-phosphate of ATP to the 2'-hydroxyl group of macrolide antibiotics."
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His205 in the ATP-binding motif is critical for catalysis (H205A retains <1% activity).
"the specific activity of the H205A mutant enzyme was reduced to less than 1% of that of the wild enzyme."
Structural Basis for Kinase-Mediated Macrolide Antibiotic Resistance
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Crystal structures of MPH(2')-II (the MphB class) were solved in the apo state and in complex with GTP analogs and six macrolides.
"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."
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Mph enzymes share the aminoglycoside-phosphotransferase (protein-kinase-like) fold but have a large interdomain linker forming an expanded antibiotic-binding pocket.
"the enzymes are related to the aminoglycoside phosphotransferases, but are distinguished from them by the presence of a large interdomain linker that contributes to an expanded antibiotic binding pocket."
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A hydrophobic, partly negatively charged binding pocket (with a conserved aspartate) rationalizes the broad-spectrum macrolide resistance.
"This pocket is largely hydrophobic, with a negatively charged patch located at a conserved aspartate residue, rationalizing the broad-spectrum resistance conferred by the enzymes."
The evolution of substrate discrimination in macrolide antibiotic resistance enzymes
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Contrary to earlier reports of a narrow range, MphB confers resistance to all macrolides tested and inactivates both telithromycin and azithromycin.
"we found that MphB confers resistance to all macrolides tested...and inactivates both telithromycin and azithromycin"
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Tandem mass spectrometry confirmed that MphB phosphorylates the desosamine 2'-OH of erythromycin.
"Using tandem mass spectrometry, we also confirmed that MphB phosphorylates the desosamine 2′-OH of erythromycin"
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MphB is one of the mobilized Mph homologs widespread in Gram-negative bacteria.
"MphA, MphB, and MphE are widespread in Gram-negative bacteria"
Genome sequence of adherent-invasive Escherichia coli and comparative genomic analysis with other E. coli pathotypes
Look and Outlook on Enzyme-Mediated Macrolide Resistance
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MPH(2')-II (MphB) inactivates 16-membered macrolides and the ketolide telithromycin in addition to 14-/15-membered macrolides, distinguishing it from the narrower MPH(2')-I (MphA).
"MPH(2′)-I can only efficiently inactivate 14- and 15-membered lactone macrolides, whereas MPH(2′)-II can additionally inactivate 16-membered lactone macrolides and the ketolide, telithromycin"