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 an ~10-fold decrease in the K(m) value of GTP and an ~320-fold increase in the K(m) value of ATP. In APH(2")-IVa, F95Y substitution results in a modest decrease in the K(m) 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.

Fig 1

Stereoviews of the nucleotide-binding pockets in APH(2″)-IIa (A) and APH(2″)-IVa (B). The enzymes are shown in ribbon representation (blue). The available binding pocket is shown as a transparent yellow surface. In both cases, the “gatekeeper” residue was excluded from the binding cavity calculation. An ATP molecule, as bound in APH(2″)-IIa, is shown as a ball-and-stick model at the bottom of each panel and was also excluded from the cavity calculations. The secondary binding pocket is indicated by a black asterisk in each panel. (A) The APH(2″)-IIa “gatekeeper” residue (M85) was mutated to tyrosine (magenta sticks) in silico and is shown in a rotamer conformation similar to that of the original methionine. The residues which line the secondary pocket (V75 and F57), along with the conserved lysine residue (K42), are shown as cyan sticks. The rotamer conformation of a tyrosine at position 85 directed away from the ATP-binding pocket and into this putative secondary pocket is shown as white sticks. (B) The APH(2″)-IVa “gatekeeper” residue (F95) was mutated to tyrosine (magenta sticks) in silico. The secondary pocket, bounded by V78 and V61 (cyan), is able to accommodate both the wild-type phenylalanine (not shown) and the tyrosine mutant (magenta sticks).