Gretchen Hagen 3 (GH3) acyl acid amidosynthetases are encoded by early auxin-responsive genes and catalyze an ATP-dependent biosynthesis of IAA-amino acid conjugates. An amide conjugate of IAA, indole-3-acetyl-aspartate (IAA-aspartate, IAA-Asp), is a predominant form of bound auxin in immature seeds of pea. However, there is some evidence that IAA is also able to form high molecular weight amide conjugates with proteins in pea and other plant species. In this short study we report that recombinant PsGH3 IAA-amino acid synthetase, which exhibits a preference for the formation of IAA-Asp, can also conjugate IAA with the protein fraction from immature seeds of pea (S-10 fraction). We studied [14C]IAA incorporation to the S-10 protein fraction by two assays: TLC method and protein precipitation by trichloroacetic acid (TCA). In both cases, radioactivity of [14C]IAA in the protein fraction increases in comparison to the control (without PsGH3), about 9.3- and 3.17-fold, respectively. l-Asp, as a preferred substrate in the IAA conjugation catalyzed by PsGH3, down-regulates [14C]IAA conjugation to the proteins as shown by the TLC assay (∼2.8-fold decrease) and the TCA precipitation variant (∼2-fold decrease). Moreover, l-Trp that competes with Asp for the catalytic site of PsGH3 and inhibits activity of the enzyme, diminished radioactivity of [14C]IAA-proteins about 1.2- and 2.8-fold, respectively. Taking into account that amino group of an amino acid or a protein acts as an acceptor of the indole-3-acetyl moiety from IAA-AMP intermediate during GH3-dependent conjugation, we masked amine groups (α- and ε-NH2) of the S-10 protein fraction from pea seeds by reductive alkylation. The alkylated proteins revealed about 3- and 2.8-fold lower radioactivity of [14C]IAA than non-alkylated fraction for TLC and TCA precipitation variant, respectively. This is a first study demonstrating that formation of high molecular weight IAA conjugates with proteins is catalyzed by a GH3 acyl acid amidosynthetase.

中文翻译：

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豌豆 GH3 酰基酸酰胺合成酶将 IAA 与豌豆未成熟种子中的蛋白质结合——形成生长素高分子量结合物的新视角


Gretchen Hagen 3 (GH3) 酰基酸酰胺合成酶由早期生长素响应基因编码，催化 IAA-氨基酸缀合物的 ATP 依赖性生物合成。 IAA 的酰胺缀合物，吲哚-3-乙酰基-天冬氨酸（IAA-天冬氨酸，IAA-Asp）是未成熟豌豆种子中结合生长素的主要形式。然而，有一些证据表明 IAA 也能够与豌豆和其他植物物种中的蛋白质形成高分子量酰胺缀合物。在这项简短的研究中，我们报道了重组 PsGH3 IAA-氨基酸合成酶，它表现出对 IAA-Asp 形成的偏好，也可以将 IAA 与来自豌豆未成熟种子的蛋白质部分（S-10 部分）结合。我们通过两种测定法研究了 [14C]IAA 掺入 S-10 蛋白组分：TLC 方法和三氯乙酸 (TCA) 蛋白沉淀法。在这两种情况下，与对照（不含 PsGH3）相比，蛋白质级分中 [14C]IAA 的放射性分别增加约 9.3 倍和 3.17 倍。 L-Asp 作为 PsGH3 催化的 IAA 缀合中的首选底物，下调 [14C]IAA 与蛋白质的缀合，如 TLC 测定（下降约 2.8 倍）和 TCA 沉淀变体（下降约 2 倍）所示减少）。此外，L-Trp 与 Asp 竞争 PsGH3 的催化位点并抑制酶的活性，使 [14C]IAA 蛋白的放射性分别降低约 1.2 倍和 2.8 倍。考虑到氨基酸或蛋白质的氨基在 GH3 依赖性缀合过程中充当 IAA-AMP 中间体吲哚-3-乙酰基部分的受体，我们掩盖了氨基酸或蛋白质的氨基（α-和 ε-NH2）通过还原烷基化从豌豆种子中提取 S-10 蛋白质。烷基化蛋白质显示大约 3- 和 2。对于 TLC 和 TCA 沉淀变体，[14C]IAA 的放射性分别比非烷基化组分低 8 倍。这是第一项研究证明高分子量 IAA 与蛋白质的结合物是由 GH3 酰基酸酰胺合成酶催化形成的。