page_title: Kanamycin kinase
section_title: Mechanism
hierarchical_section_title: Kanamycin kinase / Mechanism
caption_attribution_description: English: APH ligands ATP and kanamycin with relevant active site residues shown
context_page_description: Aminoglycoside-3'-phosphotransferase, also known as aminoglycoside kinase, is an enzyme that primarily catalyzes the addition of phosphate from ATP to the 3'-hydroxyl group of a 4,6-disubstituted aminoglycoside, such as kanamycin. However, APH has also been found to phosphorylate at the 5'-hydroxyl group in 4,5-disubstituted aminoglycosides, which lack a 3'-hydroxyl group, and to diphosphorylate hydroxyl groups in aminoglycosides that have both 3'- and 5'-hydroxyl groups. Primarily positively charged at biological conditions, aminoglycosides bind to the negatively charged backbone of nucleic acids to disrupt protein synthesis, effectively inhibiting bacterial cell growth. APH mediated phosphorylation of aminoglycosides effectively disrupts their mechanism of action, introducing a phosphate group that reduces their binding affinity due to steric hindrances and unfavorable electrostatic interactions. APH is primarily found in certain species of gram-positive bacteria.
  This enzyme belongs to the family of transferases, specifically those transferring phosphorus-containing groups with an alcohol group as acceptor.
context_section_description: While earlier studies of APH(3') supported a mechanism involving the nucleophilic attack of γ-phosphate by the 3'-hydroxyl, more recent studies suggest that APH(3’) catalyzes the transfer of the γ-phosphate from ATP to an aminoglycoside through a dissociative mechanism, where deprotonation of the substrate is not critical to phosphate transfer, but instead the stabilization of a metaphosphate transition state.
  Additionally, APH(3’) has a nucleotide positioning loop (NPL) that closes down on the enzyme active site after binding ATP, facilitating the phosphorylation of the 3’-hydroxyl group. Key to correctly positioning the phosphate group are Ser27 and Met26 residues. Initially, two magnesium ions stabilized by Asn195 and Asp208 facilitate the binding of ATP in the active site and orient the ß- and γ-phosphate groups. The NPL then undergoes a conformational change to form a hydrogen bond between Ser27 and the ß-phosphate group. Upon binding of substrate, APH(3') undergoes another conformational change to orient Ser27 such that its amide backbone disrupts the alignment of ß-phosphate and γ-phosphate, weakening the γ-phosphate bond. The amide backbone of Met26 forms a hydrogen bond with the metaphosphate to stabilize the transition state, as a magnesium ion (designated Mg1) then lengthens the γ-phosphate bond, breaking it and effectively phosphorylating the hydroxyl group.
caption: ADP and Kanamycin A in the active site of APH(3'). Two magnesium ions are coordinated by Asn195 and Asp208 residues, which in turn facilitate the binding of ATP in the active site. The NPL, in conjunction with magnesium ions, mediate the phosphorylation of aminoglycosides.
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