Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA.
Antimicrob Agents Chemother. 2013 Aug;57(8):3763-6. doi: 10.1128/AAC.00381-13. Epub 2013 May 28.
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.
氨基糖苷 2″-磷酸转移酶 APH(2")-IIa 和 APH(2")-IVa 可以利用 ATP 和 GTP 作为共底物，因为这两种酶都具有重叠但不同的 ATP 和 GTP 结合结构模板。APH(2″)-IIIa 仅使用 GTP，因为其 ATP 结合模板被一个大的酪氨酸“守门员”残基所阻断。APH(2")-IIa 和 APH(2")-IVa 中的“守门员”残基 M85 和 F95 分别被酪氨酸取代，不会显著改变酶产生的抗生素敏感性谱。在 APH(2")-IIa 中，M85Y 取代导致 GTP 的 K(m) 值降低约 10 倍，ATP 的 K(m) 值增加约 320 倍。在 APH(2")-IVa 中，F95Y 取代导致 GTP 和 ATP 的 K(m) 值略有降低。结构分析表明，在 APH(2")-IIa M85Y 突变体中，酪氨酸阻断了 ATP 进入结合位点的正确位置，而 APH(2")-IVa F95Y 突变体的较大核苷三磷酸 (NTP) 结合口袋允许酪氨酸移动，从而使 ATP 结合模板能够进入。
