Mechanism of aminoglycoside antibiotic kinase APH(3')-IIIa: role of the nucleotide positioning loop

UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology

Publication Date


Document Type



Adenosine Triphosphate; Amino Acid Sequence; Aminoglycosides; Binding Sites; Kanamycin Kinase; Kinetics; Methionine; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Nucleotides; Phosphorus; Serine; Substrate Specificity


Biochemistry | Enzymes and Coenzymes | Medicinal-Pharmaceutical Chemistry | Therapeutics


The aminoglycoside antibiotic resistance kinases (APHs) and the Ser/Thr/Tyr protein kinases share structural and functional homology but very little primary sequence conservation ( < 5%). A region of structural, but not amino acid sequence, homology is the nucleotide positioning loop (NPL) that closes down on the enzyme active site upon binding of ATP. This loop region has been implicated in facilitating phosphoryl transfer in protein kinases; however, there is no primary sequence conservation between APHs and protein kinases in the NPL. There is an invariant Ser residue in all APH NPL regions, however. This residue in APH(3')-IIIa (Ser27), an enzyme widespread in aminoglycoside-resistant Enterococci, Streptococci, and Staphylococci, directly interacts with the beta-phosphate of ATP through the Ser hydroxymethyl group and the amide hydrogen in the 3D structure of the enzyme. Mutagenesis of this residue to Ala and Pro supported a role for the Ser amide hydrogen in nucleotide capture and phosphoryl transfer. A molecular model of the proposed dissociative transition state, which is consistent with all of the available mechanistic data, suggested a role for the amide of the adjacent Met26 in phosphoryl transfer. Mutagenesis studies confirmed the importance of the amide hydrogen and suggest a mechanism where Ser27 anchors the ATP beta-phosphate facilitating bond breakage with the gamma-phosphate during formation of the metaphosphate-like transition, which is stabilized by interaction with the amide hydrogen of Met26. The APH NPL therefore acts as a lever, promoting phosphoryl transfer to the aminoglycoside substrate, with the biological outcome of clinically relevant antibiotic resistance.

DOI of Published Version



Biochemistry. 2002 Jun 4;41(22):7001-7. doi:10.1021/bi0256680

Journal/Book/Conference Title



At the time of publication, Paul Thompson was not yet affiliated with UMass Medical School.

Related Resources

Link to Article in PubMed