Novel <i>α</i> ‐KG/Fe(II)‐Dependent Dioxygenases Catalyzing C1 <i>β</i> ‐Hydroxylation and Construction of 5/7/6‐Skeleton of Highly Oxygenated Taxoids

Here, we report the discovery and functional characterization of one novel taxane C1β-hydroxylase (TmT1βH), belonging to the α-ketoglutarate (α-KG)/Fe(II)-dependent dioxygenase family from Taxus × media cell cultures. The incubation of recombinant TmT1βH with 1β-dehydroxybaccatin IV (1) as a substrate led to the production of a major C1-hydroxylated product, baccatin IV (1a), and a minor product, 15-hydroxy-11(15→1)abeo-baccatin IV (1b), a non-classical 5/7/6-type taxane. Moreover, in vitro biochemical assays, molecular docking, and molecular dynamics simulation combined with site-directed mutagenesis revealed the critical amino acid residues for TmT1βH catalysis. Substrate specificity investigations revealed that TmT1βH preferred taxoids with high oxygenation level. Notably, we have also discovered a novel specific enzyme (Tm576) belonging to α-KG/Fe(II)-dependent dioxygenase that was able to convert 1 to 1b independently. A mechanism that the 5/7/6-membered carbon framework arises from prototypical 6/8/6-type taxane skeleton via radical rearrangement was proposed based on DFT calculations. More importantly, we artificially reconstructed the biosynthetic pathway of two important taxanes, baccatin IV, and baccatin VI, from GGPP in tobacco system. This work not only fully characterizes the role of C1β-hydroxylase of taxoids, but also offered new insights into the formation of taxane structural diversity.