Molecular mechanism of aminoglycoside antibiotic kinase APH(3′)-IIIa: Roles of conserved active site residues

The aminoglycoside antibiotic kinases (APHs) constitute a clinically important group of antibiotic resistance enzymes. APHs share structural and functional homology with Ser/Thr and Tyr kinases, yet only five amino acids are invariant between the two groups of enzymes and these residues are all located within the nucleotide binding regions of the proteins. We have performed site-directed mutagenesis on all five conserved residues in the aminoglycoside kinase APH(3′)-IIIa: Lys⁴⁴ and Glu⁶⁰ involved in ATP capture, a putative active site base required for deprotonating the incoming aminoglycoside hydroxyl group Asp¹⁹⁰, and the Mg ²⁺ ligands Asn¹⁹⁵ and Glu²⁰⁸, which coordinate two Mg²⁺ ions, Mg1 and Mg2. Previous structural and mutagenesis evidence have demonstrated that Lys⁴⁴ interacts directly with the phosphate groups of ATP; mutagenesis of invariant Glu⁶⁰, which forms a salt bridge with the ε-amino group of Lys⁴⁴, demonstrated that this residue does not play a critical role in ATP recognition or catalysis. Results of mutagenesis of Asp¹⁹⁰ were consistent with a role in proper positioning of the aminoglycoside hydroxyl during phosphoryl transfer but not as a general base. The Mg1 and Mg2 ligand Asp²⁰⁸ was found to be absolutely required for enzyme activity and the Mg2 ligand Asn ¹⁹⁵ is important for Mg·ATP recognition. The mutagenesis results together with solvent isotope, solvent viscosity, and divalent cation requirements are consistent with a dissociative mechanism of phosphoryl transfer where initial substrate deprotonation is not essential for phosphate transfer and where Mg2 and Asp²⁰⁸ likely play a critical role in stabilization of a metaphosphate-like transition state. These results lay the foundation for the synthesis of transition state mimics that could reverse aminoglycoside antibiotic resistance in vivo.