Cryptic and Stereospecific Hydroxylation, Oxidation, and Reduction in Platensimycin and Platencin Biosynthesis.

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, alpha-hydroxyl, beta-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 alpha-hydroxylation in PTM and PTN biosynthesis. Combining in vitro and in vivo studies, we show that PtmO3 and PtmO6 are two functionally redundant alpha-ketoglutarate-dependent dioxygenases that generate a cryptic C7 beta-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 beta-hydroxyl to alpha-hydroxyl via a C7 ketone intermediate. PtmO3 and PtmO6 represent the first dedicated C7 beta-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.