Department of Microbiology, University of Georgia, Athens, Georgia, USA.
Department of Microbiology, University of Georgia, Athens, Georgia, USA
Appl Environ Microbiol. 2019 Mar 6;85(6). doi: 10.1128/AEM.03029-18. Print 2019 Mar 15.
Acylation of epsilon amino groups of lysyl side chains is a widespread modification of proteins and small molecules in cells of all three domains of life. Recently, we showed that and encode the GCN5-related -acetyltransferase (GNAT) SatA that can acetylate and inactivate streptothricin, which is a broad-spectrum antibiotic produced by actinomycetes in the soil. To determine functionally relevant residues of SatA (SatA), a mutational screen was performed, highlighting the importance of a conserved area near the C terminus. Upon inspection of the crystal structure of the Ames SatA (SatA; PDB entry 3PP9), this area appears to form a pocket with multiple conserved aromatic residues; we hypothesized this region contains the streptothricin-binding site. Chemical and site-directed mutagenesis was used to introduce missense mutations into , and the functionality of the variants was assessed using a heterologous host (). Results of isothermal titration calorimetry experiments showed that residue Y164 of SatA was important for binding streptothricin. Results of size exclusion chromatography analyses showed that residue D160 was important for dimerization. Together, these data advance our understanding of how SatA interacts with streptothricin. This work provides insights into how an abundant antibiotic found in soil is bound to the enzyme that inactivates it. This work identifies residues for the binding of the antibiotic and probes the contributions of substituting side chains for those in the native protein, providing information regarding hydrophobicity, size, and flexibility of the antibiotic binding site.
ε 氨基酰化赖氨酸侧链是所有三个生命领域的细胞中蛋白质和小分子的广泛修饰方式。最近，我们表明 和 编码 GCN5 相关的 -乙酰转移酶（GNAT）SatA，它可以乙酰化和失活链霉素，链霉素是土壤中放线菌产生的广谱抗生素。为了确定 SatA（SatA）的功能相关残基，进行了突变筛选，突出了靠近 C 末端的保守区域的重要性。在检查 Ames SatA（SatA；PDB 条目 3PP9）的晶体结构时，该区域似乎形成了一个带有多个保守芳香族残基的口袋；我们假设该区域包含链霉素结合位点。使用化学和定点诱变将错义突变引入 ，并使用异源宿主（）评估变体的功能。等温滴定量热实验的结果表明，SatA 的残基 Y164 对结合链霉素很重要。尺寸排阻色谱分析的结果表明，残基 D160 对二聚化很重要。这些数据共同提高了我们对 SatA 如何与链霉素相互作用的理解。这项工作深入了解了土壤中发现的丰富抗生素是如何与使抗生素失活的酶结合的。这项工作确定了抗生素结合的残基，并探测了用天然蛋白质中的侧链取代这些残基的贡献，提供了有关抗生素结合位点疏水性、大小和灵活性的信息。
