Hybrid natural products are compounds that originate from diverse biosynthetic pathways and undergo a conjugation process, which enables them to expand their chemical diversity and biological functionality. Terpene-amino acid meroterpenoids have garnered increasing attention in recent years, driven by the discovery of noteworthy examples such as the anthelmintic CJ-12662, the insecticidal paeciloxazine, and aculene A (1). In the biosynthesis of terpene-amino acid natural products, single module nonribosomal peptide synthetases (NRPSs) have been identified to involve in the esterification step, catalyzing the fusion of modified terpene and amino acid components. Despite prior investigations into these NRPSs through gene deletion or in vivo experiments, the enzymatic basis and mechanistic insights underlying this family of single-module NRPSs remain unclear. In this study, we performed biochemical characterization of AneB by in vitro characterization, molecular docking and site-directed mutagenesis. The enzyme reaction analyses, performed with L-proline and daucane/nordaucane sesquiterpene substrates, revealed that AneB specifically esterifies the C10-OH of aculenes with L-proline. Notably, in contrast to ThmA in CJ-12662 biosynthesis, which exclusively recognizes oxygenated amorpha-4,11-diene sesquiterpenes for L-tryptophan transfer, AneB demonstrates broad substrate selectivity, including oxygenated amorpha-4,11-diene and 2-phenylethanol, resulting in the production of diverse unnatural prolyl compounds. Furthermore, site-directed mutagenesis experiments indicated the involvement of H794 and D798 in the esterification catalyzed by AneB. Lastly, domain swapping between AneB and ThmA unveiled that the A‒T domains of ThmA can be effectively harnessed by the C domain of AneB for L-tryptophan transfer, thus highlighting the potential of the C domain of AneB for generating various terpene-amino acid meroterpenoid derivatives.

中文翻译：

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真菌单模块非核糖体肽合成酶在萜烯-氨基酸类萜生物合成中的表征和催化研究

混合天然产物是源自不同生物合成途径并经历结合过程的化合物，这使得它们能够扩展其化学多样性和生物功能。近年来，由于驱虫药 CJ-12662、杀虫剂哌西洛嗪和 aculene A 等值得注意的例子的发现，萜烯-氨基酸类半萜类化合物受到越来越多的关注 (1)。在萜烯-氨基酸天然产物的生物合成中，单模块非核糖体肽合成酶（NRPS）已被鉴定参与酯化步骤，催化修饰萜烯和氨基酸组分的融合。尽管之前通过基因删除或体内实验对这些 NRPS 进行了研究，但该单模块 NRPS 家族的酶学基础和机制见解仍不清楚。在这项研究中，我们通过体外表征、分子对接和定点诱变对 AneB 进行了生化表征。使用 L-脯氨酸和胡萝卜烷/正胡萝卜烷倍半萜底物进行的酶反应分析表明，AneB 用 L-脯氨酸特异性酯化 aculenes 的 C10-OH。值得注意的是，与 CJ-12662 生物合成中的 ThmA 不同，ThmA 专门识别用于 L-色氨酸转移的氧化紫穗槐-4,11-二烯倍半萜，AneB 表现出广泛的底物选择性，包​​括氧化紫穗槐-4,11-二烯和 2-苯基乙醇，导致产生多种非天然脯氨酰化合物。此外，定点诱变实验表明H794和D798参与AneB催化的酯化反应。最后，AneB 和 ThmA 之间的结构域交换揭示了 ThmA 的 A-T 结构域可以被 AneB 的 C 结构域有效利用进行 L-色氨酸转移，从而凸显了 AneB 的 C 结构域生成各种萜氨基酸的潜力类萜衍生物。