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, 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.