Notes: mphB (MphB, macrolide 2'-phosphotransferase II; UniProt A0A0H3EUF3)

Identity

• UniProt: A0A0H3EUF3 (TrEMBL, unreviewed), 302 aa, ~34.5 kDa.
• Organism: Escherichia coli O83:H1 strain NRG 857C (adherent-invasive E. coli, AIEC). NCBITaxon:685038.
• Encoded on plasmid pO83_CORR (OrderedLocusName NRG857_30123). Mobile/plasmid-borne resistance gene.
• UniProt SubName: "Macrolide 2'-phosphotransferase II protein MphB".
• CARD/ARO: ARO:3000318 (mphB); resistance mechanism = antibiotic inactivation [DR line in uniprot.txt].
• NCBIfam HMM: NF000242 (macrolide_MphB) — a curated family HMM specifically for MphB.
• RefSeq WP_000031017.1 (widely distributed, conserved sequence).

Domain / fold

• Pfam PF01636 (APH, aminoglycoside phosphotransferase) domain, aa 23–265.
• PANTHER PTHR21310 (Aminoglycoside Phosphotransferase Enzymes), subfamily SF15.
• CDD cd05152 (MPH2).
• Gene3D 3.90.1200.10 + 3.30.200.20 (Phosphorylase Kinase domain 1); SUPFAM SSF56112 Protein kinase-like (PK-like).
• => Protein-kinase-like (ePK/APH) fold; this is the structural superfamily shared by aminoglycoside
  phosphotransferases and Ser/Thr/Tyr protein kinases. MphB is a small-molecule (macrolide) kinase, NOT a
  protein kinase and NOT an aminoglycoside kinase despite the family name.

Function (well established in literature)

• MphB = macrolide 2'-phosphotransferase II (MPH(2')II). Transfers the γ-phosphate of a purine nucleoside
  triphosphate to the 2'-OH of macrolide antibiotics, producing the inactive macrolide 2'-O-phosphate. This
  detoxifies/inactivates the antibiotic and confers macrolide resistance.
  PMID:10428938] transfers the gamma phosphate of ATP to the 2'-OH group of macrolide antibiotics.")
• Reaction matches GO:0050073 "macrolide 2'-kinase activity": ATP + oleandomycin = ADP + 2 H+ + oleandomycin 2'-O-phosphate.
• Inactivated product confirmed as oleandomycin 2'-phosphate by TLC.
  PMID:1330822

Substrate / cofactor specificity

• Broad macrolide substrate range: active on BOTH 14-membered (e.g. oleandomycin, erythromycin) AND
  16-membered (e.g. spiramycin, tylosin, josamycin) ring macrolides; also 15-membered. mphB confers
  high-level resistance to spiramycin (16-membered) when expressed in S. aureus.
  PMID:1330822
  PMID:9503630
• Phosphate donor: purine nucleotides ITP, GTP and ATP all effective as cofactors.
  PMID:1330822
• Divalent metal cation involvement: activity affected by iodine, EDTA, or divalent cations (EDTA inhibition
  is consistent with a Mg2+-dependent phosphotransfer, as for the protein-kinase-like fold).
  PMID:1330822
• Constitutive, intracellular enzyme; pI 5.3, optimum pH 8.2, optimum temp 40°C.
  PMID:1330822

Catalytic residues (site-directed mutagenesis)

• Conserved aspartates in the ATP-binding/catalytic region are essential:
  D200, D209, D219, D231 — alanine substitution abolishes oleandomycin inactivation.
  D227A retains ~7% activity (non-essential; involved in recognizing 16-membered macrolides).
  PMID:10428938
  (D200 proposed as catalytic base activating the 2'-OH; D219/D231 implicated in ATP binding — consistent with
  the protein-kinase-like fold catalytic/Mg2+-binding aspartates.)

mphA vs mphB

• Two distinct enzymes in E. coli: mphA (type I, MPH(2')I) and mphB (type II, MPH(2')II). Both phosphorylate
  the macrolide 2'-OH. mphA is inducible and is regulated by a repressor mphR(A); mphB (this gene) is
  constitutive. mphB has broad activity across 14/15/16-membered macrolides.
  PMID:9503630

GOA status

• QuickGO GOA file is EMPTY (header only) for this TrEMBL accession → existing_annotations starts empty.
• The only electronic GO term on the UniProt record is GO:0016740 "transferase activity" (IEA:UniProtKB-KW),
  which is far too general. The correct specific MF is GO:0050073 (macrolide 2'-kinase activity).

GO terms to assign (NEW)

• MF: GO:0050073 macrolide 2'-kinase activity (exact; supported by all biochemical refs).
• BP: GO:0046677 response to antibiotic (resistance/detoxification). Possibly GO:0017001 antibiotic catabolic
  process — but phosphorylation inactivates (modifies) rather than catabolizes/degrades the macrolide, so
  "response to antibiotic" is the safer/standard resistance BP; antibiotic catabolic process is arguable.
• MF supporting: GO:0016301 kinase activity / phosphotransferase; ATP binding (GO:0005524) — but uses
  ITP/GTP too, so a narrow ATP-binding term is not ideal; the specific GO:0050073 already captures the activity.

References (PMIDs cached)

• PMID:21108814 — AIEC NRG857C genome (source of this sequence/accession).
• PMID:1330822 — Purification & characterization of MPH(2')II (Kono et al. 1992). KEY biochemistry.
• PMID:9503630 — mphB expression in S. aureus; 16-membered macrolide (spiramycin) resistance (Noguchi 1998).
• PMID:10428938 — Functional amino acids (catalytic Asp residues) of MPH(2')II (Taniguchi 1999). KEY mechanism.

Update: falcon deep research (2026-06-12) — additional references incorporated

Falcon deep research completed (genes/ECO8N/mphB/mphB-deep-research-falcon.md, 24 citations)
and surfaced three additional high-value primary papers, now cached and added to the review:

• PMID:28416110 — Fong et al. 2017, Structure. "Structural Basis for Kinase-Mediated Macrolide Antibiotic
  Resistance." First crystal structures of MPH(2')-I and MPH(2')-II (the MphB class), apo and with GTP analogs
• six macrolides. Confirms the bi-lobed APH/protein-kinase-like fold with a large interdomain linker forming
  an expanded, hydrophobic macrolide-binding pocket (conserved aspartate negative patch) → rationalizes broad
  spectrum. Structures captured with GTP analogs (GTP a preferred donor in vitro).
  PMID:28416110-II in the apo state, and in complex with GTP analogs and six different macrolides.")
• PMID:29317655 — Pawlowski et al. 2018, Nat Commun (PMC5760710, full text). "The evolution of substrate
  discrimination in macrolide antibiotic resistance enzymes." Mass-spec confirms MphB phosphorylates the
  desosamine 2'-OH of erythromycin; revises older narrow-spectrum view — MphB inactivates azithromycin AND
  telithromycin and confers resistance to all macrolides tested. MphB widespread/mobilized in Gram-negatives.
  PMID:29317655
  PMID:29317655
• PMID:15033229 — Taniguchi et al. 2004, FEMS Microbiol Lett. "The role of histidine residues conserved in the
  putative ATP-binding region of macrolide 2'-phosphotransferase II." His205 critical (H205A <1% activity).
  PMID:15033229

Net effect on review: description strengthened (broad spectrum incl. azithromycin/telithromycin; crystal
structures; GTP preference; His205). GO calls unchanged (GO:0050073 MF, GO:0046677 BP) — now multiply
supported. suggested_questions/experiments revised since the structure (Fong 2017) and broad-spectrum
question (Pawlowski 2018) are now answered.

Note on providers: perplexity not configured in this container (only openai/falcon); openai API key invalid
(401). falcon's wrapper reported a 600s timeout but the Edison run actually completed and wrote the report +
artifacts. just is not installed here, so underlying uv run ai-gene-review ... commands were used directly.

Update: CARD ARO:3000318 (mphB) — curated AMR data incorporated

CARD entry https://card.mcmaster.ca/ARO:3000318 (AMR Gene Family: Macrolide phosphotransferase (MPH);
mechanism: antibiotic inactivation; Protein Homolog Model, BLASTP bitscore cutoff 600). Curated drug list:
erythromycin, roxithromycin, clarithromycin, dirithromycin, oleandomycin (14-membered), azithromycin
(15-membered), spiramycin, tylosin (16-membered), and the ketolide telithromycin. Mechanism: phosphorylation
at the 2'-OH of the desosamine sugar of 14- and 16-membered macrolides. Two CARD-cited PMIDs newly added:

• PMID:8900063 — Noguchi et al. 1996, FEMS Microbiol Lett. Original cloning/sequencing of mphB. KEY identity
  anchor: encodes a 302-aa / 34483-Da protein, matching this UniProt entry (302 aa, 34485 MW) exactly.
  PMID:8900063
• PMID:17302923 — Chesneau et al. 2007, FEMS Microbiol Lett. Resistance phenotypes of mph genes in an
  efflux-deficient E. coli AG100A host. mphB confers spiramycin + telithromycin resistance; in this older
  study azithromycin resistance was attributed uniquely to mphA — the narrow-spectrum view of MphB later
  overturned by Pawlowski 2018 (PMID:29317655).
  PMID:17302923
  PMID:17302923

Net effect: identity now triple-anchored (UniProt SubName + CARD ARO:3000318 + cloning paper exact 302aa/
34483Da match + NCBIfam NF000242 macrolide_MphB HMM). Description substrate range expanded to the full
curated CARD drug list and the historical narrow→broad spectrum reassessment documented. GO calls unchanged.