Plants rely on concentration gradients of the native auxin, indole-3-acetic acid (IAA), to modulate plant growth and development. Both metabolic and transport processes participate in the dynamic regulation of IAA homeostasis. Free IAA levels can be reduced by inactivation mechanisms, such as conjugation and degradation. IAA can be conjugated via ester linkage to glucose, or via amide linkage to amino acids, and degraded via oxidation. Members of the UDP glucosyl transferase (UGT) family catalyze the conversion of IAA to indole-3-acetyl-1-glucosyl ester (IAGlc); by contrast, IAA is irreversibly converted to indole-3-acetyl-l-aspartic acid (IAAsp) and indole-3-acetyl glutamic acid (IAGlu) by Group II of the GRETCHEN HAGEN3 (GH3) family of acyl amido synthetases. Dioxygenase for auxin oxidation (DAO) irreversibly oxidizes IAA to oxindole-3-acetic acid (oxIAA) and, in turn, oxIAA can be further glucosylated to oxindole-3-acetyl-1-glucosyl ester (oxIAGlc) by UGTs. These metabolic pathways have been identified based on mutant analyses, in vitro activity measurements, and in planta feeding assays. In vitro assays for studying protein activity are based on producing Arabidopsis enzymes in a recombinant form in bacteria or yeast followed by recombinant protein purification. However, the need to extract and purify the recombinant proteins represents a major obstacle when performing in vitro assays. In this work we report a rapid, reproducible and cheap method to screen the enzymatic activity of recombinant proteins that are known to inactivate IAA. The enzymatic reactions are carried out directly in bacteria that produce the recombinant protein. The enzymatic products can be measured by direct injection of a small supernatant fraction from the bacterial culture on ultrahigh-performance liquid chromatography coupled to electrospray ionization tandem spectrometry (UHPLC–ESI-MS/MS). Experimental procedures were optimized for testing the activity of different classes of IAA-modifying enzymes without the need to purify recombinant protein. This new method represents an alternative to existing in vitro assays. It can be applied to the analysis of IAA metabolites that are produced upon supplementation of substrate to engineered bacterial cultures and can be used for a rapid screening of orthologous candidate genes from non-model species.

中文翻译：

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一种用于快速筛选 IAA 分解代谢酶的细菌测定法

植物依靠天然生长素、吲哚-3-乙酸 (IAA) 的浓度梯度来调节植物的生长和发育。代谢和运输过程都参与了 IAA 稳态的动态调节。游离 IAA 水平可以通过失活机制降低，例如结合和降解。IAA 可以通过酯键与葡萄糖结合，或通过酰胺键与氨基酸结合，并通过氧化降解。UDP 葡糖基转移酶 (UGT) 家族的成员催化 IAA 转化为吲哚-3-乙酰基-1-葡糖基酯 (IAGlc)；相比之下，IAA 被 GRETCHEN HAGEN3 (GH3) 酰基酰胺合成酶家族的 II 组不可逆地转化为吲哚-3-乙酰基-l-天冬氨酸 (IAAsp) 和吲哚-3-乙酰谷氨酸 (IAGlu)。用于生长素氧化 (DAO) 的双加氧酶不可逆地将 IAA 氧化为 oxindole-3-乙酸 (oxIAA)，而 oxIAA 又可以通过 UGT 进一步葡糖基化为 oxindole-3-acetyl-1-glucosyl ester (oxIAGlc)。这些代谢途径已根据突变分析、体外活性测量和足底喂养试验进行了鉴定。用于研究蛋白质活性的体外测定基于在细菌或酵母中以重组形式产生拟南芥酶，然后进行重组蛋白质纯化。然而，在进行体外检测时，需要提取和纯化重组蛋白是一个主要障碍。在这项工作中，我们报告了一种快速、可重复且廉价的方法来筛选已知可使 IAA 失活的重组蛋白的酶活性。酶促反应直接在产生重组蛋白的细菌中进行。酶促产物可以通过在超高效液相色谱与电喷雾电离串联光谱法 (UHPLC-ESI-MS/MS) 上直接注入细菌培养物中的少量上清液部分来测量。优化了实验程序以测试不同类别的 IAA 修饰酶的活性，而无需纯化重组蛋白。这种新方法代表了现有体外测定的替代方法。它可用于分析在向工程细菌培养物中补充底物后产生的 IAA 代谢物，并可用于快速筛选来自非模型物种的直系同源候选基因。酶促产物可以通过在超高效液相色谱与电喷雾电离串联光谱法 (UHPLC-ESI-MS/MS) 上直接注入细菌培养物中的少量上清液部分来测量。优化了实验程序以测试不同类别的 IAA 修饰酶的活性，而无需纯化重组蛋白。这种新方法代表了现有体外测定的替代方法。它可用于分析在向工程细菌培养物中补充底物后产生的 IAA 代谢物，并可用于快速筛选来自非模型物种的直系同源候选基因。酶促产物可以通过在超高效液相色谱与电喷雾电离串联光谱法 (UHPLC-ESI-MS/MS) 上直接注入细菌培养物中的少量上清液部分来测量。优化了实验程序以测试不同类别的 IAA 修饰酶的活性，而无需纯化重组蛋白。这种新方法代表了现有体外测定的替代方法。它可用于分析在向工程细菌培养物中补充底物后产生的 IAA 代谢物，并可用于快速筛选来自非模型物种的直系同源候选基因。