Benzoic acid‐derived compounds, such as polyprenylated benzophenones and xanthones, attract the interest of scientists due to challenging chemical structures and diverse biological activities. The genus Hypericum is of high medicinal value, as exemplified by H. perforatum. It is rich in benzophenone and xanthone derivatives, the biosynthesis of which requires the catalytic activity of benzoate‐coenzyme A (benzoate‐CoA) ligase (BZL), which activates benzoic acid to benzoyl‐CoA. Despite remarkable research so far done on benzoic acid biosynthesis in planta, all previous structural studies of BZL genes and proteins are exclusively related to benzoate‐degrading microorganisms. Here, a transcript for a plant acyl‐activating enzyme (AAE) was cloned from xanthone‐producing Hypericum calycinum cell cultures using transcriptomic resources. An increase in the HcAAE1 transcript level preceded xanthone accumulation after elicitor treatment, as previously observed with other pathway‐related genes. Subcellular localization of reporter fusions revealed the dual localization of HcAAE1 to cytosol and peroxisomes owing to a type 2 peroxisomal targeting signal. This result suggests the generation of benzoyl‐CoA in Hypericum by the CoA‐dependent non‐β‐oxidative route. A luciferase‐based substrate specificity assay and the kinetic characterization indicated that HcAAE1 exhibits promiscuous substrate preference, with benzoic acid being the sole aromatic substrate accepted. Unlike 4‐coumarate‐CoA ligase and cinnamate‐CoA ligase enzymes, HcAAE1 did not accept 4‐coumaric and cinnamic acids, respectively. The substrate preference was corroborated by in silico modeling, which indicated valid docking of both benzoic acid and its adenosine monophosphate intermediate in the HcAAE1/BZL active site cavity.

中文翻译：

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混杂的辅酶A连接酶为金丝桃属植物的黄酮生物合成提供苯甲酰辅酶A

由于具有挑战性的化学结构和多样的生物活性，衍生自苯甲酸的化合物，例如聚戊二烯基二苯甲酮和氧杂蒽酮，引起了科学家的兴趣。金丝桃属植物具有很高的药用价值，例如贯叶连翘。它富含二苯甲酮和黄酮衍生物，其生物合成需要苯甲酸酯辅酶A（苯甲酸酯-CoA）连接酶（BZL）的催化活性，该酶可将苯甲酸活化为苯甲酰-CoA。尽管迄今为止在植物体内苯甲酸生物合成方面进行了令人瞩目的研究，但BZL以前的所有结构研究基因和蛋白质仅与降解苯甲酸酯的微生物有关。在这里，利用转录组学资源，从生产黄酮的金丝桃金丝桃细胞培养物中克隆了植物酰基激活酶（AAE）的转录本。在增加Hc的AAE1转录水平之前，如前面其他途径相关基因观察诱发剂处理后，呫吨酮的积累。报告子融合的亚细胞定位显示由于2型过氧化物酶体靶向信号，Hc AAE1在细胞质和过氧化物酶体中双重定位。该结果表明金丝桃属植物中苯甲酰辅酶A的生成通过依赖CoA的非β氧化途径。基于荧光素酶的底物特异性测定和动力学表征表明Hc的AAE1表现出混杂的底物偏好，与苯甲酸被接受唯一的芳香底物。不像4-香豆酸辅酶A连接酶和肉桂酰辅酶A连接酶，Hc的AAE1没有接受4香豆和肉桂酸，分别。通过计算机模拟证实了底物的偏爱，这表明苯甲酸及其单磷酸腺苷中间体在Hc AAE1 / BZL活性位点腔中的有效对接。