Bulky "gatekeeper" residue changes the cosubstrate specificity of aminoglycoside 2-phosphotransferase IIa

Abstract

The aminoglycoside 2-phosphotransferases APH(2)-IIa and APH(2)-IVa can utilize ATP and GTP as cosubstrates, since both enzymes possess overlapping but discrete structural templates for ATP and GTP binding. APH(2)-IIIa uses GTP exclusively, because its ATP-binding template is blocked by a bulky tyrosine gatekeeper residue. Replacement of the gatekeeper residues M85 and F95 in APH(2)-IIa and APH(2)-IVa, respectively, by tyrosine does not significantly change the antibiotic susceptibility profiles produced by the enzymes. In APH(2)-IIa, M85Y substitution results in an10-fold decrease in the Km value of GTP and an320-fold increase in the Km value of ATP. In APH(2)-IVa, F95Y substitution results in a modest decrease in the Km values of both GTP and ATP. Structural analysis indicates that in the APH(2)-IIa M85Y mutant, tyrosine blocks access of ATP to the correct position in the binding site, while the larger nucleoside triphosphate (NTP)-binding pocket of the APH(2)-IVa F95Y mutant allows the tyrosine to move away, thus giving access to the ATP-binding template. Copyright © 2013, American Society for Microbiology. All Rights Reserved.