Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.
Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden
Antimicrob Agents Chemother. 2020 May 21;64(6). doi: 10.1128/AAC.00371-20.
Spectinomycin is a ribosome-binding antibiotic that blocks the translocation step of translation. A prevalent resistance mechanism is modification of the drug by aminoglycoside nucleotidyl transferase (ANT) enzymes of the spectinomycin-specific ANT(9) family or by enzymes of the dual-specificity ANT(3")(9) family, which also acts on streptomycin. We previously reported the structural mechanism of streptomycin modification by the ANT(3")(9) AadA from ANT(9) from adenylates the 9-hydroxyl of spectinomycin. Here, we present the first structures of spectinomycin bound to an ANT enzyme. Structures were solved for ANT(9) in apo form, in complex with ATP, spectinomycin, and magnesium, or in complex with only spectinomycin. ANT(9) shows an overall structure similar to that of AadA, with an N-terminal nucleotidyltransferase domain and a C-terminal α-helical domain. Spectinomycin binds close to the entrance of the interdomain cleft, while ATP is buried at the bottom. Upon drug binding, the C-terminal domain rotates 14 degrees to close the cleft, allowing contacts of both domains with the drug. Comparison with AadA shows that spectinomycin specificity is explained by a straight α helix and a shorter α-α loop, which would clash with the larger streptomycin substrate. In the active site, we observed two magnesium ions, one of them in a previously unobserved position that may activate the 9-hydroxyl for deprotonation by the catalytic base Glu-86. The observed binding mode for spectinomycin suggests that spectinamides and aminomethyl spectinomycins, recent spectinomycin analogues with expansions in position 4 of the C ring, are also subjected to modification by ANT(9) and ANT(3")(9) enzymes.
壮观霉素是一种核糖体结合抗生素，可阻断翻译的易位步骤。一种流行的耐药机制是通过壮观霉素特异性氨基糖苷核苷转移酶（ANT）酶家族的氨基糖苷核苷转移酶（ANT）或通过同时作用于链霉素的双重特异性 ANT（3"）（9）家族的酶修饰药物。我们之前报道了来自 ANT（9）的 ANT（3"）（9）AadA 修饰链霉素的结构机制，该酶使壮观霉素的 9-羟基腺嘌呤化。在这里，我们首次展示了与 ANT 酶结合的壮观霉素的结构。我们解决了 apo 形式的 ANT（9）、与 ATP、壮观霉素和镁复合的 ANT（9）以及仅与壮观霉素复合的 ANT（9）的结构。ANT（9）的整体结构与 AadA 相似，具有 N 端核苷酸转移酶结构域和 C 端α-螺旋结构域。壮观霉素靠近结构域间裂隙的入口结合，而 ATP 则埋藏在底部。药物结合后，C 端结构域旋转 14 度以关闭裂隙，使两个结构域都与药物接触。与 AadA 的比较表明，壮观霉素的特异性由直的α螺旋和较短的α-α环解释，这会与较大的链霉素底物发生冲突。在活性位点，我们观察到两个镁离子，其中一个位于以前未观察到的位置，可能通过催化碱 Glu-86 激活 9-羟基去质子化。观察到的壮观霉素结合模式表明， spectinamides 和 aminomethyl spectinomycins，最近在 C 环的 4 位扩展的壮观霉素类似物，也会被 ANT（9）和 ANT（3"）（9）酶修饰。
