Revuelta Julia, Vacas Tatiana, Torrado Mario, Corzana Francisco, Gonzalez Carlos, Jiménez-Barbero Jesús, Menendez Margarita, Bastida Agatha, Asensio Juan Luis
Instituto de Química Orgánica General (CSIC), Madrid 28006, Spain.
J Am Chem Soc. 2008 Apr 16;130(15):5086-103. doi: 10.1021/ja076835s. Epub 2008 Mar 26.
The most significant mechanism of bacterial resistance to aminoglycosides is the enzymatic inactivation of the drug. Herein, we analyze several key aspects of the aminoglycoside recognition by the resistance enzyme ANT(4') from Staphylococcus aureus, employing NMR complemented with site-directed mutagenesis experiments and measurements of the enzymatic activity on newly synthesized kanamycin derivatives. From a methodological perspective, this analysis provides the first example reported for the use of transferred NOE (trNOE) experiments in the analysis of complex molecular recognition processes, characterized by the existence of simultaneous binding events of the ligand to different regions of a protein receptor. The obtained results show that, in favorable cases, these overlapping binding processes can be isolated employing site-directed mutagenesis and then independently analyzed. From a molecular recognition perspective, this work conclusively shows that the enzyme ANT(4') displays a wide tolerance to conformational variations in the drug. Thus, according to the NMR data, kanamycin-A I/II linkage exhibits an unusual anti-Psi orientation in the ternary complex, which is in qualitative agreement with the previously reported crystallographic complex. In contrast, closely related, kanamycin-B is recognized by the enzyme in the syn-type arrangement for both glycosidic bonds. This observation together with the enzymatic activity displayed by ANT(4') against several synthetic kanamycin derivatives strongly suggests that the spatial distribution of positive charges within the aminoglycoside scaffold is the key feature that governs its preferred binding mode to the protein catalytic region and also the regioselectivity of the adenylation reaction. In contrast, the global shape of the antibiotic does not seem to be a critical factor. This feature represents a qualitative difference between the target A-site RNA and the resistance enzyme ANT(4') as aminoglycoside receptors.
细菌对氨基糖苷类药物产生耐药性的最重要机制是药物的酶促失活。在此，我们利用核磁共振（NMR）技术，并结合定点突变实验以及对新合成的卡那霉素衍生物的酶活性测定，分析了金黄色葡萄球菌耐药酶ANT(4')对氨基糖苷类药物识别的几个关键方面。从方法学角度来看，该分析首次报道了利用转移核Overhauser效应（trNOE）实验来分析复杂的分子识别过程，其特点是配体与蛋白质受体的不同区域同时发生结合事件。所得结果表明，在有利情况下，这些重叠的结合过程可通过定点突变进行分离，然后独立分析。从分子识别角度来看，这项工作确凿地表明，酶ANT(4')对药物的构象变化具有广泛的耐受性。因此，根据NMR数据，卡那霉素 - A的I/II连接在三元复合物中呈现出不寻常的反式 - Psi取向，这与先前报道的晶体复合物在定性上是一致的。相比之下，与之密切相关的卡那霉素 - B在糖苷键方面均以顺式排列被该酶识别。这一观察结果以及ANT(4')对几种合成卡那霉素衍生物所表现出的酶活性强烈表明，氨基糖苷类药物支架内正电荷的空间分布是决定其与蛋白质催化区域优先结合模式以及腺苷化反应区域选择性的关键特征。相比之下，抗生素的整体形状似乎不是关键因素。这一特征代表了作为氨基糖苷类药物受体的靶标A位点RNA与耐药酶ANT(4')之间的质的差异。
