Terpene indole alkaloids (TIAs) are plant-derived specialized metabolites with widespread use in medicine. Species-specific pathways derive various TIAs from common intermediates, strictosidine or strictosidinic acid, produced by coupling tryptamine with secologanin or secologanic acid. The penultimate reaction in this pathway is catalyzed by either secologanin synthase (SLS) or secologanic acid synthase (SLAS) according to whether plants produce secologanin from loganin or secologanic acid from loganic acid. Previous work has identified SLSs and SLASs from different species, but the determinants of selectivity remain unclear. Here, combining molecular modeling, ancestral sequence reconstruction, and biochemical methodologies, we identified key residues that toggle SLS and SLAS selectivity in two CYP72A (cytochrome P450) subfamily enzymes from Camptotheca acuminata. We found that the positions of foremost importance are in substrate recognition sequence 1 (SRS1), where mutations to either of two adjacent histidine residues switched selectivity; His131Phe selects for and increases secologanin production whereas His132Asp selects for secologanic acid production. Furthermore, a change in SRS3 in the predicted substrate entry channel (Arg/Lys270Thr) and another in SRS4 at the start of the I-helix (Ser324Glu) decreased enzyme activity toward either substrate. We propose that the Camptotheca SLASs have maintained the broadened activities found in a common asterid ancestor, even as the Camptotheca lineage lost its ability to produce loganin while the campanulid and lamiid lineages specialized to produce secologanin by acquiring mutations in SRS1. The identification here of the residues essential for the broad substrate scope of SLASs presents opportunities for more tailored heterologous production of TIAs.

萜烯吲哚生物碱 (TIA) 是植物衍生的特殊代谢物，广泛用于医学。物种特异性途径从常见的中间体、胡豆苷或胡豆苷酸衍生出各种 TIA，这些中间体是通过将色胺与 secologanin 或 secologanic 酸偶联产生的。该途径中的倒数第二个反应由 secologanin 合酶 (SLS) 或 secologanic 酸合酶 (SLAS) 催化，具体取决于植物是从马钱素产生 secologanin 还是从马钱酸产生 secologanic 酸。以前的工作已经确定了来自不同物种的 SLS 和 SLAS，但选择性的决定因素仍不清楚。在这里，结合分子建模、祖先序列重建和生化方法，我们确定了在两种 CYP72A（细胞色素 P450）亚家族酶中切换 SLS 和 SLAS 选择性的关键残基。喜树。我们发现最重要的位置在底物识别序列 1 (SRS1) 中，其中两个相邻组氨酸残基之一的突变切换了选择性；His131Phe 选择并增加 secologanin 的产生，而 His132Asp 选择 secologanic 酸的产生。此外，预测的底物进入通道 (Arg/Lys270Thr) 中 SRS3 的变化和 I-螺旋 (Ser324Glu) 开始处 SRS4 中的另一个变化降低了对任一底物的酶活性。我们建议喜树属 SLAS 保持了在一个共同的小行星祖先中发现的扩大的活动，即使喜树属谱系失去了产生马球素的能力，而钟形和拉米亚科谱系通过获得 SRS1 中的突变而专门生产 seologanin。此处对 SLAS 广泛底物范围所必需的残基的鉴定为更量身定制的 TIA 异源生产提供了机会。