| Feature/Question | Evidence summary | Key source(s) with year, URL/DOI, and Citation ID |
|---|---|---|
| Reaction catalyzed | A0A0H3EUF3 matches the characterized MphB/MPH(2')-II class: a macrolide 2'-phosphotransferase that inactivates macrolide antibiotics by O-phosphorylation. The reaction transfers a phosphate from a nucleoside triphosphate to the drug, abolishing activity. | Taniguchi et al., 2004, *FEMS Microbiology Letters*; https://doi.org/10.1016/S0378-1097(03)00961-3 (pqac-00000001, pqac-00000002); Fong et al., 2017, *Structure*; https://doi.org/10.1016/j.str.2017.03.007 (pqac-00000003) |
| Phosphorylation site | The modified position is the desosamine 2'-OH of the macrolide. Tandem-MS-supported work identified MphB as phosphorylating the desosamine 2'-OH of erythromycin, and related figures mark the same 2'-OH site on azithromycin. | Pawlowski et al., 2018, *Nature Communications*; https://doi.org/10.1038/s41467-017-02680-0 (pqac-00000000, pqac-00000005, pqac-00000006) |
| Nucleotide donor preference | The donor specificity is somewhat mixed across studies: biochemical mutagenesis assays used ATP and describe transfer of the γ-phosphate of ATP, whereas structural/enzymology work reported that MPH(2')-II shows a preference for GTP over ATP. Together, these data support that MphB is a kinase-like phosphotransferase with strong purine nucleotide usage, with GTP preference highlighted by the 2017 structural study. | Taniguchi et al., 2004; https://doi.org/10.1016/S0378-1097(03)00961-3 (pqac-00000001, pqac-00000002); Fong et al., 2017; https://doi.org/10.1016/j.str.2017.03.007 (pqac-00000003, pqac-00000007) |
| Metal/cofactor | Divalent metal is required/implicated in catalysis and nucleotide binding. Structural work observed at least one metal ion (Mg2+ or Ca2+) in the nucleotide pocket, and mutational analysis linked His205 to ATP binding via Mg-mediated interactions. | Fong et al., 2017; https://doi.org/10.1016/j.str.2017.03.007 (pqac-00000004); Taniguchi et al., 2004; https://doi.org/10.1016/S0378-1097(03)00961-3 (pqac-00000002) |
| Substrate spectrum | MphB is broad-spectrum among macrolides. Reported substrates include 14-, 15-, and 16-membered macrolides such as oleandomycin, troleandomycin, erythromycin, clarithromycin, roxithromycin, azithromycin, kitasamycin, spiramycin, josamycin, rokitamycin, tylosin, and the ketolide telithromycin; one study explicitly states MphB conferred resistance to all tested macrolides and inactivated azithromycin and telithromycin. | Taniguchi et al., 2004; https://doi.org/10.1016/S0378-1097(03)00961-3 (pqac-00000002); Fong et al., 2017; https://doi.org/10.1016/j.str.2017.03.007 (pqac-00000003, pqac-00000007); Pawlowski et al., 2018; https://doi.org/10.1038/s41467-017-02680-0 (pqac-00000000, pqac-00000005) |
| Resistance phenotype data | Expression of mphB increases macrolide resistance in bacterial hosts. Quantitatively, mutating conserved residues reduced MICs relative to wild type (H198A to about one-half and H205A to about one-eighth of wild-type MIC), and a related mphB-like plasmid ORF conferred a 32-fold increase in erythromycin resistance when expressed in *E. coli*; an additional surveillance paper cites prior *E. coli* mphB with erythromycin MIC 128 µg/mL. | Taniguchi et al., 2004; https://doi.org/10.1016/S0378-1097(03)00961-3 (pqac-00000002); Yang et al., 2014; https://doi.org/10.1128/AEM.03257-13 (pqac-00000012); Wang et al., 2023; https://doi.org/10.3389/fcimb.2023.1116172 (pqac-00000015) |
| Key catalytic residues | Functionally important residues include Asp200, His205, and Asp219 in/near the nucleotide-binding catalytic region. Mutagenesis showed H205 is critical: H205A dropped activity to <1% with oleandomycin, whereas H198A and H205N retained substantial activity, indicating H205 is especially important for catalysis/nucleotide handling. | Fong et al., 2017; https://doi.org/10.1016/j.str.2017.03.007 (pqac-00000004); Taniguchi et al., 2004; https://doi.org/10.1016/S0378-1097(03)00961-3 (pqac-00000001, pqac-00000002) |
| Structural fold/domains | MphB has a kinase-like aminoglycoside phosphotransferase-related fold with two lobes/domains. Structural studies describe an N-terminal β-sheet-rich lobe, a linker segment, and a mainly α-helical C-terminal lobe/subdomains forming a deep macrolide-binding cleft; this aligns well with the UniProt/APH-family domain assignment for A0A0H3EUF3. | Fong et al., 2017; https://doi.org/10.1016/j.str.2017.03.007 (pqac-00000003, pqac-00000004, pqac-00000008); Pawlowski et al., 2018; https://doi.org/10.1038/s41467-017-02680-0 (pqac-00000006) |
| Genetic context/mobility | mphB is mobile and has been found on transferable multidrug-resistance plasmids in *E. coli*. A transferable hybrid plasmid pAPEC-O103-ColBM carries mphB in extraintestinal *E. coli*, and broader literature/evidence links mph-family genes to plasmids, transposon-associated regions, and mosaic mobile elements, supporting horizontal spread across taxa. | Johnson et al., 2010, *Infection and Immunity*; https://doi.org/10.1128/IAI.01174-09 (pqac-00000014); Yang et al., 2014; https://doi.org/10.1128/AEM.03257-13 (pqac-00000012) |
| Real-world surveillance/WGS notes | Recent WGS-based surveillance continues to detect mphB in clinically relevant Gram-negative pathogens, although most 2023–2024 azithromycin surveillance emphasizes mphA rather than mphB. A 2024 hospital-isolate genomics study identified mphB in multidrug-resistant *Klebsiella pneumoniae*, and 2023 Salmonella plasmid surveillance cited prior mphB-associated high MICs in *E. coli* while illustrating how WGS/hybrid assembly is used to track mobile macrolide resistance determinants across Enterobacterales. | Dinda et al., 2024, *Access Microbiology*; https://doi.org/10.1099/acmi.0.000667.v4 (pqac-00000015); Wang et al., 2023; https://doi.org/10.3389/fcimb.2023.1116172 (pqac-00000015) |


*Table: This table summarizes the experimentally supported functional annotation of UniProt A0A0H3EUF3 as MphB/macrolide 2'-phosphotransferase type II. It compiles reaction chemistry, substrate range, catalytic features, structural biology, mobility, and recent surveillance relevance using only the cited evidence contexts.*