Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, 1414 Cumberland Avenue, Knoxville, Tennessee 37996, United States.
Biochemistry. 2012 Nov 13;51(45):9147-55. doi: 10.1021/bi301126g. Epub 2012 Nov 2.
The aminoglycoside nucleotidyltransferase (4') (ANT) is an aminoglycoside-modifying enzyme that detoxifies antibiotics by nucleotidylating at the C4'-OH site. Previous crystallographic studies show that the enzyme is a homodimer and each subunit binds one kanamycin and one Mg-AMPCPP, where the transfer of the nucleotidyl group occurs between the substrates bound to different subunits. In this work, sedimentation velocity analysis of ANT by analytical ultracentrifugation showed the enzyme exists as a mixture of a monomer and a dimer in solution and that dimer formation is driven by hydrophobic interactions between the subunits. The binding of aminoglycosides shifts the equilibrium toward dimer formation, while the binding of the cosubstrate, Mg-ATP, has no effect on the monomer-dimer equilibrium. Surprisingly, binding of several divalent cations, including Mg(2+), Mn(2+), and Ca(2+), to the enzyme also shifted the equilibrium in favor of dimer formation. Binding studies, performed by electron paramagnetic resonance spectroscopy, showed that divalent cations bind to the aminoglycoside binding site in the absence of substrates with a stoichiometry of 2:1. Energetic aspects of binding of all aminoglycosides to ANT were determined by isothermal titration calorimetry to be enthalpically favored and entropically disfavored with an overall favorable Gibbs energy. Aminoglycosides in the neomycin class each bind to the enzyme with significantly different enthalpic and entropic contributions, while those of the kanamycin class bind with similar thermodynamic parameters.
氨基糖苷核苷转移酶（4'）（ANT）是一种氨基糖苷修饰酶，通过在 C4'-OH 位点核苷酰化来解毒抗生素。先前的晶体学研究表明，该酶是一个同源二聚体，每个亚基结合一个卡那霉素和一个 Mg-AMPCPP，其中核苷基团的转移发生在结合于不同亚基的底物之间。在这项工作中，通过分析超速离心对 ANT 的沉降速度分析表明，该酶在溶液中以单体和二聚体的混合物形式存在，并且二聚体形成是由亚基之间的疏水相互作用驱动的。氨基糖苷的结合将平衡向二聚体形成方向移动，而共底物 Mg-ATP 的结合对单体-二聚体平衡没有影响。令人惊讶的是，几种二价阳离子，包括 Mg(2+)、Mn(2+)和 Ca(2+)，与酶的结合也有利于二聚体的形成。通过电子顺磁共振波谱进行的结合研究表明，二价阳离子在没有底物的情况下以 2:1 的化学计量结合到氨基糖苷结合位点。通过等温滴定量热法确定了所有氨基糖苷与 ANT 结合的能量学方面，结果表明结合是焓有利且熵不利的，总体上具有有利的吉布斯自由能。新霉素类的氨基糖苷各自与酶结合具有显著不同的焓和熵贡献，而卡那霉素类的则具有相似的热力学参数。
