Department of Chemistry, California Lutheran University, Thousand Oaks, CA, United States.
Department of Chemistry, California Lutheran University, Thousand Oaks, CA, United States.
Methods Enzymol. 2024;702:75-87. doi: 10.1016/bs.mie.2024.06.017. Epub 2024 Jul 20.
NIS Synthetases are a widely distributed, novel superfamily of enzymes critical to stealth siderophore production-small molecules increasingly associated with virulence. Study of these enzymes for inhibition or utilization in biosynthesis of new antibiotics has been hindered by multiple kinetics assays utilizing different limiting reporters or relying on product dissociation as a precursor to signal. We present a label free, continuous readout assay optimized for NIS Synthetase systems utilizing an isothermal titration calorimetry instrument. This assay has been tested in an iterative system comparing multiple turnovers on a single substrate to a single bond formation event and is able to delineate these complex kinetics well. The ITC-based kinetic assay is the first label-free assay for the NIS field, which may allow for more detailed kinetic comparisons in the future, and may also have broader use for iterative enzymes in general.
NIS 合酶是一个广泛分布的新型酶超家族，对隐蔽性铁载体的产生至关重要，而这些小分子与毒力的关系日益密切。由于利用不同的限制报告物或依赖于产物解离作为信号前体的多种动力学测定，这些酶在抑制或用于新抗生素生物合成方面的研究受到了阻碍。我们提出了一种无标记、连续读取的测定方法，该方法针对利用等温滴定量热法仪器的 NIS 合酶系统进行了优化。该测定方法已在一个迭代系统中进行了测试，该系统将单个底物上的多次周转与单个键形成事件进行了比较，并且能够很好地区分这些复杂的动力学。基于 ITC 的动力学测定方法是 NIS 领域的第一个无标记测定方法，它可能允许在未来进行更详细的动力学比较，并且可能对一般的迭代酶具有更广泛的用途。
