Functional characterization and structural bases of two class I diterpene synthases in pimarane-type diterpene biosynthesis

Abstract

Pimarane-type diterpenoids are widely distributed in all domains of life, but no structures or catalytic mechanisms of pimarane-type diterpene synthases (DTSs) have been characterized. Here, we report that two class I DTSs, Sat1646 and Stt4548, each accept copalyl diphosphate (CPP) as the substrate to produce isopimara-8,15-diene (1). Sat1646 can also accept syn-CPP and produce syn-isopimaradiene/pimaradiene analogues (2–7), among which 2 possesses a previously unreported "6/6/7" ring skeleton. We solve the crystal structures of Sat1646, Sat1646 complexed with magnesium ions, and Stt4548, thereby revealing the active sites of these pimarane-type DTSs. Substrate modeling and subsequent site-directed mutagenesis experiments demonstrate different structural bases of Sat1646 and Stt4548 for 1 production. Comparisons with previously reported DTSs reveal their distinct carbocation intermediate stabilization mechanisms, which control the conversion of a single substrate CPP into structurally diverse diterpene products. These results illustrate the structural bases for enzymatic catalyses of pimarane-type DTSs, potentially facilitating future DTS engineering and combinatorial biosynthesis.

Crystal Structure Based Mutagenesis of Cattleyene Synthase Leads to the Generation of Rearranged Polycyclic Diterpenes

“…and Stt4548 from Streptomyces sp. (Figure 1A) [27] . Structural knowledge of DTSs is of interest to deepen our mechanistic understanding of these enzymes and allows for structure based site‐directed mutagenesis [25, 28, 29] …”

“…(Figure 1A). [27] Structural knowledge of DTSs is of interest to deepen our mechanistic understanding of these enzymes and allows for structure based site-directed mutagenesis. [25,28,29] We recently discovered the cattleyene (1) synthase (CyS) from Streptomyces cattleya (Figure 1B) and studied its cyclization mechanism through isotopic labelings.…”

20-nor-Isopimarane and isopimarane diterpenoids produced by Aspergillus sp. WT03

“…9. 14,20,21 Compound 9 was derived from geranylgeranyl diphosphate (GGPP) which underwent cyclization, 22 deprotonation of H-14, oxidation and dehydrogenation of 14-OH. On the one hand, the decarboxylation of 9 at C-20, subsequent hydroxylation at C-6 and C-10, and dehydration resulted in the formation of 7 .…”

Kristallstrukturbasierte Mutagenese der Cattleyene‐Synthase führt zur Bildung umgelagerter polycyclischer Diterpene

“…und Stt4548 aus Streptomyces sp. (Abbildung 1A) [27] . Das strukturelle Wissen über DTSs ist von Interesse, um unser mechanistisches Verständnis dieser Enzyme zu vertiefen, und ermöglicht eine strukturbasierte ortsgerichtete Mutagenese [25, 28, 29] …”

“…(Abbildung 1A). [27] Das strukturelle Wissen über DTSs ist von Interesse, um unser mechanistisches Verständnis dieser Enzyme zu vertiefen, und ermöglicht eine strukturbasierte ortsgerichtete Mutagenese. [25,28,29] Kürzlich entdeckten wir die Cattleyen (1)-Synthase (CyS) aus Streptomyces cattleya (Abbildung 1B) und untersuchten ihren Cyclisierungsmechanismus durch Isotopenmarkierung.…”