title: Molecular determinants of antibiotic recognition and resistance by aminoglycoside phosphotransferase (3')-IIIa: a calorimetric and mutational analysis.
abstract: The growing threat from the emergence of multidrug resistant pathogens highlights a critical need to expand our currently available arsenal of broad-spectrum antibiotics. In this connection, new antibiotics must be developed that exhibit the abilities to circumvent known resistance pathways. An important step toward achieving this goal is to define the key molecular interactions that govern antibiotic resistance. Here, we use site-specific mutagenesis, coupled with calorimetric, NMR, and enzymological techniques, to define the key interactions that govern the binding of the aminoglycoside antibiotics neomycin and kanamycin B to APH(3')-IIIa (an antibiotic phosphorylating enzyme that confers resistance). Our mutational analyses identify the D261, E262, and C-terminal F264 residues of the enzyme as being critical for recognition of the two drugs as well as for the manifestation of the resistance phenotype. In addition, the E160 residue is more important for recognition of kanamycin B than neomycin, with mutation of this residue partially restoring sensitivity to kanamycin B but not to neomycin. By contrast, the D193 residue partially restores sensitivity to neomycin but not to kanamycin B, with the origins of this differential effect being due to the importance of D193 for catalyzing the phosphorylation of neomycin. These collective mutational results, coupled with (15)N NMR-derived pK(a) and calorimetrically derived binding-linked drug protonation data, identify the 1-, 3-, and 2'-amino groups of both neomycin and kanamycin B as being critical functionalities for binding to APH(3')-IIIa. These drug amino functionalities represent potential sites of modification in the design of next-generation compounds that can overcome APH(3')-IIIa-induced resistance.
authors.affiliation: Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854-5635, USA.
authors.forename: Malvika
authors.initials: M
authors.lastname: Kaul
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Barbieri
authors.forename: Annankoil R
authors.initials: AR
authors.lastname: Srinivasan
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Pilch
mesh_terms: D000596:Amino Acids; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D002151:Calorimetry; D002384:Catalysis / Q000187:drug effects; D002942:Circular Dichroism; D003067:Coenzymes / Q000378:metabolism; D004252:DNA Mutational Analysis; D024881:Drug Resistance, Bacterial / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007612:Kanamycin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D019868:Kanamycin Kinase / Q000378:metabolism*; D007613:Kanamycin Resistance; D009682:Magnetic Resonance Spectroscopy; D008826:Microbial Sensitivity Tests; D009154:Mutation / Q000235:genetics; D009355:Neomycin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011522:Protons; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics; D017186:Titrimetry
chemical_list: D000596:Amino Acids; D000900:Anti-Bacterial Agents; D003067:Coenzymes; D011522:Protons; C001495:bekanamycin; D007612:Kanamycin; D019868:Kanamycin Kinase; D009355:Neomycin