Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, Illinois, USA.
Department of Cell and Developmental Biology, University of Illinois Urbana-Champaign, Urbana, Illinois, USA; Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, Illinois, USA; Department of Plant Biology, University of Illinois Urbana-Champaign, Urbana, Illinois, USA.
J Biol Chem. 2022 Sep;298(9):102237. doi: 10.1016/j.jbc.2022.102237. Epub 2022 Jul 6.
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.
萜吲哚生物碱（TIAs）是植物来源的具有广泛医学用途的特殊代谢产物。特定物种的途径从共同的中间体——斯特罗金或斯特罗金酸——衍生出各种 TIAs，该中间体由色胺与_secologanin_或_secologanic_酸偶联而成。该途径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_loganin_产生_secologanin 还是从_loganic_酸产生_secologanic 酸。以前的工作已经从不同的物种中鉴定出了 SLS 和 SLAS，但选择性的决定因素仍不清楚。在这里，我们结合分子建模、祖先序列重建和生化方法，鉴定了来自喜树的两个 CYP72A（细胞色素 P450）亚家族酶中切换 SLS 和 SLAS 选择性的关键残基。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的突变会改变选择性；His131Phe 选择并增加 secologanin 的产生，而 His132Asp 选择 secologanic 酸的产生。此外，预测底物进入通道中的 SRS3 发生变化（Arg/Lys270Thr）和 I 螺旋起始处的 SRS4 发生变化（Ser324Glu）会降低对任一底物的酶活性。我们提出，喜树 SLAS 保留了在一个共同的 Asterid 祖先中发现的广泛活性，即使喜树谱系失去了产生_loganin 的能力，而 campanulid 和 lamiid 谱系通过在 SRS1 中获得突变而专门产生 secologanin。这里鉴定出的对 SLAS 广泛底物范围至关重要的残基为更有针对性的 TIAs 异源生产提供了机会。
