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

Abstract

The crystal structures of cattleyene synthase (apo-CyS), and CyS complexed with geranylgeranyl pyrophosphate (GGPP) were solved. The CyS C59A variant exhibited an increased production of cattleyene and other diterpenes with diverse skeletons. Its structure showed a widened active site cavity explaining the relaxed selectivity. Isotopic labeling experiments revealed a remarkable cyclization mechanism involving several skeletal rearrangements for one of the novel diterpenes.

Decoding Catalysis by Terpene Synthases

“…In the C59A variant, the increased active site volume improves GGPP uptake but at the expense of product selectivity, by diminishing the cation−π interactions with the substrate. 118 Since many bioactive and industrially relevant terpenoids are polycyclic, there is a need to understand cyclization in detail. The studies and results presented in this section are, therefore, fundamental to the future rational engineering of TSs.…”

Functional Characterization and Cyclization Mechanism of a Diterpene Synthase Catalyzing the Skeleton Formation of Cephalotane‐Type Diterpenoids

“…Chemie (15,16, C 3 )GGPP by CsCTS yielded the product, (15,16,17-13 C 3 )-1, with an increased molecular mass of m/z 275 along with strongly enhanced NMR resonance signals for C15, C16, and C17 due to substitution with 13 C. Four weak doublets (J = 33.0 Hz, 34.5 Hz, 33.0 Hz, and 34.5 Hz) with C2, C7, C10 and C14, respectively, in the 13 C NMR spectrum (Figure S16) were observed because of the direct CÀ 13 C bond. (18,18,18-2 H 3 )GGPP was also enzymatically converted with CsCTS, and the NMR signals for H18 and C18 of the product (18,18,18-2 H 3 )-1 vanished as a result of the incorporation of 2 H (Figures S17 and S18).…”

Functional Characterization and Cyclization Mechanism of a Diterpene Synthase Catalyzing the Skeleton Formation of Cephalotane‐Type Diterpenoids

“…M1-M4 were the deprotonated products of carbocation intermediates A + , B + , D + , and H + , respectively, which support the proposed cyclization mechanism. (15,16, C 3 )GGPP by CsCTS yielded the product, (15,16,17-13 C 3 )-1, with an increased molecular mass of m/z 275 along with strongly enhanced NMR resonance signals for C15, C16, and C17 due to substitution with 13 C. Four weak doublets (J = 33.0 Hz, 34.5 Hz, 33.0 Hz, and 34.5 Hz) with C2, C7, C10 and C14, respectively, in the 13 C NMR spectrum (Figure S16) were observed because of the direct CÀ 13 C bond. (18,18,18-2 H 3 )GGPP was also enzymatically converted with CsCTS, and the NMR signals for H18 and C18 of the product (18,18,18-2 H 3 )-1 vanished as a result of the incorporation of 2 H (Figures S17 and S18).…”

“…[8] Accordingly, the cyclization mechanisms of different diTPSs have fascinated chemists for decades, resulting in recent progress in unraveling the cyclization mechanisms of several diTPSs using isotopic labeling, computational chemistry, and even structural biology experiments. [9][10][11][12][13][14][15][16] However, it remains a challenge to decipher the specific cyclization cascades that form structurally unique and complex diterpene frameworks with multiple fused rings and stereogenic centers, especially those from plants.…”