Identification and characterization of key enzymes associated with cell wall biosynthesis and modification is fundamental to gain insights into cell wall dynamics. However, it is a challenge that activity assays of glycosyltransferases are very low throughput and acceptor substrates are generally not available. We optimized and validated microscale thermophoresis (MST) to achieve high throughput screening for glycosyltransferase substrates. MST is a powerful method for the quantitative analysis of protein–ligand interactions with low sample consumption. The technique is based on the motion of molecules along local temperature gradients, measured by fluorescence changes. We expressed glycosyltransferases as YFP-fusion proteins in tobacco and optimized the MST method to allow the determination of substrate binding affinity without purification of the target protein from the cell lysate. The application of this MST method to the β-1,4-galactosyltransferase AtGALS1 validated the capability to screen both nucleotide-sugar donor substrates and acceptor substrates. We also expanded the application to members of glycosyltransferase family GT61 in sorghum for substrate screening and function prediction. This method is rapid and sensitive to allow determination of both donor and acceptor substrates of glycosyltransferases. MST enables high throughput screening of glycosyltransferases for likely substrates, which will narrow down their in vivo function and help to select candidates for further studies. Additionally, this method gives insight into biochemical mechanism of glycosyltransferase function.

中文翻译：

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微尺度热泳作为筛选参与细胞壁生物合成的糖基转移酶的有力工具。

鉴定和表征与细胞壁生物合成和修饰相关的关键酶是深入了解细胞壁动力学的基础。然而，糖基转移酶活性测定的通量非常低并且受体底物通常不可用是一个挑战。我们优化并验证了微型热泳 (MST) 以实现糖基转移酶底物的高通量筛选。MST 是一种以低样品消耗量定量分析蛋白质-配体相互作用的强大方法。该技术基于分子沿局部温度梯度的运动，通过荧光变化测量。我们在烟草中将糖基转移酶表达为 YFP 融合蛋白，并优化了 MST 方法，以允许在不从细胞裂解物中纯化目标蛋白的情况下测定底物结合亲和力。将此 MST 方法应用于 β-1,4-半乳糖基转移酶 AtGALS1 验证了筛选核苷酸-糖供体底物和受体底物的能力。我们还将应用扩展到高粱中糖基转移酶家族 GT61 的成员，用于底物筛选和功能预测。该方法快速且灵敏，可以测定糖基转移酶的供体和受体底物。MST 能够对糖基转移酶进行高通量筛选以寻找可能的底物，这将缩小它们的体内功能并有助于选择用于进一步研究的候选物。此外，