The Thermodynamics of Ligand Binding to the Aminoglycoside O- Nucleotidyltransferase(4′) and Variants Yields Clues about Thermophilic Properties
The Thermodynamics of Ligand Binding to the Aminoglycoside O- Nucleotidyltransferase(4′) and Variants Yields Clues about Thermophilic Properties
复制标题
配体与氨基糖苷 O-核苷酸转移酶 (4-2) 和变体结合的热力学提供了有关嗜热特性的线索
DOI：
10.1021/acs.biochem.8b01201
复制
发表时间：
2019
期刊：
影响因子：
2.9
通讯作者：
Serpersu, Engin H.
中科院分区：
文献类型：
--
作者：
Kocaman, Seda;Serpersu, Engin H.
氨基糖苷类核苷转移酶（4′）是一种底物高度混杂的酶，催化AMP基团从ATP转移到许多结构不同的氨基糖苷类的4′-OH位点，这导致其作为抗生素的有效性消失。两个携带单位点突变的热稳定变体用于确定与嗜热性相关的分子特性。酶-配体相互作用的热力学表明，一种变体（T130 K）具有与嗜温野生型（WT）相同的性质，而另一种变体（D80 Y）则表现不同。D80 Y和T130 K/WT对之间的差异包括热容（ΔCp）的变化，这取决于D80 Y的温度，而不是WT或T130 K。D80 Y的Δ Cp随温度的变化（ΔΔCp）仅取决于H2O中的氨基糖苷类，并且与D2 O中的所有氨基糖苷类保持相同。此外，偏移温度（Toff），即在H2O和D2 O中产生相同的温度差，随着WT和T130 K温度的增加而变大，但对于D80 Y几乎保持不变。在H2O和D2 O的研究表明，溶剂重组成为配体结合的主要贡献者与WT和T130 K的温度增加，但低频振动模式的变化是D80 Y的主要贡献者。本文中提出的数据表明，与酶-配体相互作用，如配体结合的热力学，全球性能，可能会产生嗜热性的线索，并允许我们区分那些只是一个更热稳定的版本的嗜温蛋白的变体。
The aminoglycoside nucleotidyltransferase(4′) is an enzyme with high substrate promiscuity and catalyzes the transfer of the AMP group from ATP to the 4′-OH site of many structurally diverse aminoglycosides, which results in the elimination of their effectiveness as antibiotics. Two thermostable variants carrying single-site mutations are used to determine the molecular properties associated with thermophilicity. The thermodynamics of enzyme–ligand interactions showed that one variant (T130K) has properties identical to those of the mesophilic wild type (WT) while the other (D80Y) behaved differently. Differences between D80Y and the T130K/WT pair include the change in heat capacity (ΔCp), which is dependent on temperature for D80Y but not for WT or T130K. The change in ΔCpwith temperature (ΔΔCp) with D80Y is dependent on aminoglycoside only in H2O and remains the same with all aminoglycosides in D2O. Furthermore, the offset temperature (Toff), the temperature difference that yields identical enthalpies in H2O and D2O, becomes larger with an increase in temperature for WT and T130K but remains mostly unchanged for D80Y. Studies in H2O and D2O revealed that solvent reorganization becomes the major contributor to ligand binding with an increase in temperature for WT and T130K, but changes in low-frequency vibrational modes are the main contributors with D80Y. Data presented in this paper suggest that global properties associated with the enzyme–ligand interactions, such as the thermodynamics of ligand binding, may yield clues about thermophilicity and permit us to distinguish those variants that are simply a more thermostable version of the mesophilic protein.