J Am Chem Soc. 2019 Mar 6;141(9):4043-4050. doi: 10.1021/jacs.8b13452. Epub 2019 Feb 19.
Platensimycin (PTM) and platencin (PTN) are highly functionalized bacterial diterpenoids of ent-kauranol and ent-atiserene biosynthetic origin. C7 oxidation in the B-ring plays a key biosynthetic role in generating structural complexity known for ent-kaurane and ent-atisane derived diterpenoids. While all three oxidation patterns, α-hydroxyl, β-hydroxyl, and ketone, at C7 are seen in both the ent-kaurane and ent-atisane derived diterpenoids, their biosynthetic origins remain largely unknown. We previously established that PTM and PTN are produced by a single biosynthetic machinery, featuring cryptic C7 oxidations at the B-rings that transform the ent-kauranol and ent-atiserene derived precursors into the characteristic PTM and PTN scaffolds. Here, we report a three-enzyme cascade affording C7 α-hydroxylation in PTM and PTN biosynthesis. Combining in vitro and in vivo studies, we show that PtmO3 and PtmO6 are two functionally redundant α-ketoglutarate-dependent dioxygenases that generate a cryptic C7 β-hydroxyl on each of the ent-kauranol and ent-atiserene scaffolds, and PtmO8 and PtmO1, a pair of NAD/NADPH-dependent dehydrogenases, subsequently work in concert to invert the C7 β-hydroxyl to α-hydroxyl via a C7 ketone intermediate. PtmO3 and PtmO6 represent the first dedicated C7 β-hydroxylases characterized to date and, together with PtmO8 and PtmO1, provide an account for the biosynthetic origins of all three C7 oxidation patterns that may shed light on other B-ring modifications in bacterial, plant, and fungal diterpenoid biosynthesis. Given their unprecedented activities in C7 oxidations, PtmO3, PtmO6, PtmO8, and PtmO1 enrich the growing toolbox of novel enzymes that could be exploited as biocatalysts to rapidly access complex diterpenoid natural products.
盘菌素 (PTM) 和盘烯素 (PTN) 是高度功能化的细菌二萜，具有 ent-贝壳杉烷和 ent-阿替色烯生物合成来源。B 环中的 C7 氧化在生成已知的 ent-贝壳杉烷和 ent-阿替色烯衍生二萜的结构复杂性方面发挥着关键的生物合成作用。虽然在 ent-贝壳杉烷和 ent-阿替色烯衍生的二萜中都可以看到 C7 上的三种氧化模式，即 α-羟基、β-羟基和酮，但它们的生物合成来源在很大程度上仍然未知。我们之前已经确定 PTM 和 PTN 是由单一的生物合成机制产生的，该机制在 B 环中具有隐蔽的 C7 氧化，将 ent-贝壳杉烷和 ent-阿替色烯衍生的前体转化为特征性的 PTM 和 PTN 支架。在这里，我们报告了一个三酶级联反应，该反应在 PTM 和 PTN 生物合成中提供 C7α-羟化。通过体外和体内研究相结合，我们表明 PtmO3 和 PtmO6 是两种功能上冗余的 α-酮戊二酸依赖性双加氧酶，它们在每个 ent-贝壳杉烷和 ent-阿替色烯支架上生成一个隐蔽的 C7β-羟基，而 PtmO8 和 PtmO1 是一对 NAD/NADPH 依赖性脱氢酶，随后协同工作，通过 C7 酮中间体将 C7β-羟基转化为α-羟基。PtmO3 和 PtmO6 代表迄今为止表征的第一个专用 C7β-羟化酶，与 PtmO8 和 PtmO1 一起，为所有三种 C7 氧化模式的生物合成起源提供了一个解释，这可能为细菌、植物和真菌二萜生物合成中的其他 B 环修饰提供启示。鉴于它们在 C7 氧化中前所未有的活性，PtmO3、PtmO6、PtmO8 和 PtmO1 丰富了不断增长的新型酶工具包，这些酶可以作为生物催化剂被利用，快速获得复杂的二萜天然产物。
