title: The Streptomyces-produced antibiotic fosfomycin is a promiscuous substrate for archaeal isopentenyl phosphate kinase.
abstract: Isopentenyl phosphate kinase (IPK) catalyzes the phosphorylation of isopentenyl phosphate to form the isoprenoid precursor isopentenyl diphosphate in the archaeal mevalonate pathway. This enzyme is highly homologous to fosfomycin kinase (FomA), an antibiotic resistance enzyme found in a few strains of Streptomyces and Pseudomonas whose mode of action is inactivation by phosphorylation. Superposition of Thermoplasma acidophilum (THA) IPK and FomA structures aligns their respective substrates and catalytic residues, including H50 and K14 in THA IPK and H58 and K18 in Streptomyces wedmorensis FomA. These residues are conserved only in the IPK and FomA members of the phosphate subdivision of the amino acid kinase family. We measured the fosfomycin kinase activity of THA IPK [K(m) = 15.1 ± 1.0 mM, and k(cat) = (4.0 ± 0.1) × 10⁻² s⁻¹], resulting in a catalytic efficiency (k(cat)/K(m) = 2.6 M⁻¹ s⁻¹) that is 5 orders of magnitude lower than that of the native reaction. Fosfomycin is a competitive inhibitor of IPK (K(i) = 3.6 ± 0.2 mM). Molecular dynamics simulation of the IPK·fosfomycin·MgATP complex identified two binding poses for fosfomycin in the IP binding site, one of which results in a complex analogous to the native IPK·IP·ATP complex that engages H50 and the lysine triangle formed by K5, K14, and K205. The other binding pose leads to a dead-end complex that engages K204 near the IP binding site to bind fosfomycin. Our findings suggest a mechanism for acquisition of FomA-based antibiotic resistance in fosfomycin-producing organisms.
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City, Utah, United States.
authors.forename: Mark F
authors.initials: MF
authors.lastname: Mabanglo
authors.forename: Adrian W R
authors.initials: AW
authors.lastname: Serohijos
authors.forename: C Dale
authors.initials: CD
authors.lastname: Poulter
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D030562:Databases, Protein; D024881:Drug Resistance, Bacterial / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005578:Fosfomycin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008968:Molecular Conformation; D056004:Molecular Dynamics Simulation; D010766:Phosphorylation / Q000187:drug effects; D010770:Phosphotransferases / Q000737:chemistry / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D055550:Protein Stability; D013302:Streptomyces / Q000201:enzymology; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013822:Thermoplasma / Q000201:enzymology*
chemical_list: D000900:Anti-Bacterial Agents; D019843:Archaeal Proteins; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D005578:Fosfomycin; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D011494:Protein Kinases; C402428:isopentenyl monophosphate kinase; D008274:Magnesium