GRETCHEN HAGEN 3 (GH3) acyl acid amido synthetases conjugate amino acids to acyl acid hormones to either activate or inactivate the hormone molecule. The largest subgroup of GH3 proteins modify the growth-promoting hormone auxin (indole-3-acetic acid; IAA) with the second largest class activating the defense hormone jasmonic acid (JA). The two-step reaction mechanism of GH3 proteins provides a potential proofreading mechanism to ensure fidelity of hormone modification. Examining pyrophosphate release in the first-half reaction of Arabidopsis GH3 proteins that modify IAA (AtGH3.2/YDK2, AtGH3.5/WES1, AtGH3.17/VAS2), JA (AtGH3.11/JAR1), and other acyl acids (AtGH3.7, AtGH3.12/PBS3) indicates that acyl acid-AMP intermediates are hydrolyzed into acyl acid and AMP in the absence of the amino acid, a typical feature of pre-transfer editing mechanisms. Single-turnover kinetic analysis of AtGH3.2/YDK2 and AtGH3.5/WES1 shows that non-cognate acyl acid-adenylate intermediates are more rapidly hydrolyzed than the cognate IAA-adenylate. In contrast, AtGH3.11/JAR1 only adenylates JA, not IAA. While some of the auxin-conjugating GH3 proteins in Arabidopsis (, AtGH3.5/WES1) accept multiple acyl acid substrates, others, like AtGH3.2/YDK2, are specific for IAA; however, both these proteins share similar active site residues. Biochemical analysis of chimeric variants of AtGH3.2/YDK2 and AtGH3.5/WES1 indicates that the C-terminal domain contributes to selection of cognate acyl acid substrates. These findings suggest that the hydrolysis of non-cognate acyl acid-adenylate intermediates, or proofreading, proceeds a slowed structural switch that provides a checkpoint for fidelity before the full reaction proceeds.

中文翻译：

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拟南芥 GH3 酰酰胺合成酶催化植物激素修饰的保真度


GRETCHEN HAGEN 3 (GH3) 酰基酰胺合成酶将氨基酸与酰基酸激素结合，从而激活或灭活激素分子。 GH3 蛋白的最大亚类可修饰生长激素生长素（吲哚-3-乙酸；IAA），第二大类可激活防御激素茉莉酸 (JA)。 GH3蛋白的两步反应机制提供了一种潜在的校对机制，以确保激素修饰的保真度。检查修饰 IAA（AtGH3.2/YDK2、AtGH3.5/WES1、AtGH3.17/VAS2）、JA（AtGH3.11/JAR1）和其他酰基酸的拟南芥 GH3 蛋白前半反应中的焦磷酸盐释放（ AtGH3.7、AtGH3.12/PBS3)表明酰基酸-AMP中间体在没有氨基酸的情况下水解成酰基酸和AMP，这是预转移编辑机制的典型特征。 AtGH3.2/YDK2 和 AtGH3.5/WES1 的单周转动力学分析表明，非同源酰基酸-腺苷酸中间体比同源 IAA-腺苷酸中间体水解得更快。相比之下，AtGH3.11/JAR1 仅腺苷酸化 JA，而不是 IAA。虽然拟南芥中的一些生长素结合 GH3 蛋白 (AtGH3.5/WES1) 接受多种酰基酸底物，但其他蛋白（如 AtGH3.2/YDK2）对 IAA 具有特异性；然而，这两种蛋白质具有相似的活性位点残基。 AtGH3.2/YDK2 和 AtGH3.5/WES1 嵌合变体的生化分析表明，C 端结构域有助于选择同源酰基酸底物。这些发现表明，非同源酰基酸-腺苷酸中间体的水解或校对会进行缓慢的结构转换，从而在整个反应进行之前提供保真度的检查点。