Document Detail

Molecular determinants of antibiotic recognition and resistance by aminoglycoside phosphotransferase (3')-IIIa: a calorimetric and mutational analysis.
MedLine Citation:
PMID:  17418235     Owner:  NLM     Status:  MEDLINE    
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.
Malvika Kaul; Christopher M Barbieri; Annankoil R Srinivasan; Daniel S Pilch
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2007-03-15
Journal Detail:
Title:  Journal of molecular biology     Volume:  369     ISSN:  0022-2836     ISO Abbreviation:  J. Mol. Biol.     Publication Date:  2007 May 
Date Detail:
Created Date:  2007-04-30     Completed Date:  2007-07-02     Revised Date:  2014-09-24    
Medline Journal Info:
Nlm Unique ID:  2985088R     Medline TA:  J Mol Biol     Country:  England    
Other Details:
Languages:  eng     Pagination:  142-56     Citation Subset:  IM    
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MeSH Terms
Amino Acids
Anti-Bacterial Agents / chemistry,  metabolism*,  pharmacology
Catalysis / drug effects
Circular Dichroism
Coenzymes / metabolism
DNA Mutational Analysis
Drug Resistance, Bacterial* / drug effects
Escherichia coli / drug effects
Hydrogen-Ion Concentration / drug effects
Kanamycin / analogs & derivatives*,  chemistry,  metabolism,  pharmacology
Kanamycin Kinase / metabolism*
Kanamycin Resistance
Magnetic Resonance Spectroscopy
Microbial Sensitivity Tests
Mutation / genetics
Neomycin / chemistry,  metabolism*,  pharmacology
Protein Binding / drug effects
Structure-Activity Relationship
Grant Support
5T32 GM08319/GM/NIGMS NIH HHS; CA097123/CA/NCI NIH HHS; F32 AI062041-02/AI/NIAID NIH HHS; F32 AI62041/AI/NIAID NIH HHS; R01 CA097123-04/CA/NCI NIH HHS; T32 GM008319/GM/NIGMS NIH HHS; T32 GM008319-15/GM/NIGMS NIH HHS
Reg. No./Substance:
0/Amino Acids; 0/Anti-Bacterial Agents; 0/Coenzymes; 0/Protons; 1404-04-2/Neomycin; 15JT14C3GI/bekanamycin; 59-01-8/Kanamycin; EC Kinase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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