4.6 Article

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

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COMMUNICATIONS CHEMISTRY
卷 4, 期 1, 页码 -

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NATURE PORTFOLIO
DOI: 10.1038/s42004-021-00578-z

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资金

  1. National Key Research and Development Program of China [2019YFC0312502]
  2. National Natural Science Foundation of China [22077007, 21877002, 81991525, 22107007, 81673332]
  3. key project at central government level: the ability establishment of sustainable use for valuable Chinese medicine resources [2060302-1903-03]
  4. China Postdoctoral Science Foundation [2019M660362]

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In this study, the structures and functions of pimarane-type diterpene synthases were characterized, revealing that Sat1646 and Stt4548 utilize distinct modes of intermediate stabilization. By comparing the mechanisms of different DTSs, it was inferred that they control the conversion of a single substrate CPP into structurally diverse diterpene products.
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. Pimaranes are diterpenoid natural products with poorly characterised biosynthetic pathways. Here pimaranetype diterpenoid synthases are functionally and structurally characterised and shown to exploit distinct modes of intermediate stabilisation.

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