Revision of the Cyclisation Mechanism for the Diterpene Spiroviolene and Investigations of Its Mass Spectrometric Fragmentation

Abstract

The diterpene spiroviolene, its diterpene synthase from Streptomyces violens and the experimentally determined terpene cyclisation mechanism were reported in 2017. Recently, the structure of spiroviolene was revised based on a total synthesis, with consequences for the cyclisation mechanism. Herein, a reinvestigation of the terpene cyclisation to spiroviolene and the mass spectrometric fragmentation mechanism investigated by 13C‐labelling experiments are presented.

Engineering of Ancestors as a Tool to Elucidate Structure, Mechanism, and Specificity of Extant Terpene Cyclase

“…Taken together, our results can explain how both extant and reconstructed ancestral SvS can act as dual functioning sesqui-/diterpene cyclases. The described diterpene reaction mechanism 4 , 58 involves major charge rearrangements ( Figure 4 ), which requires aromatic residues to sequentially stabilize the intermediate cations via π-interactions on both walls of the binding pocket (such as Trp79, Trp82, Phe84, and Trp156 on helices C/F and Phe59 and Trp308 on helices B/K), whereas the sesquiterpene reaction mechanism occurs in a more focused region of the active site. Interestingly, the tryptophan corresponding to Trp308 that we propose to be involved in the diterpene formation, is conserved in SdS and HecS, which are both sesquiterpene cyclases, even though a direct involvement of this tryptophan in the sesquiterpene reaction mechanism has been excluded.…”

“…Visual inspection shows that formation of 7′ from 6 would be feasible. Alternative mechanistic steps for the generation of 7′ from 3 were also very recently proposed …”

“…Alternative mechanistic steps for the generation of 7′ from 3 were also very recently proposed. 58 In the same manner, snapshots of the FPP conversion to (2Z,6E)-hedycaryol in SvS-A2 and SvS-WT were generated. The snapshots for SvS-A2 are shown in Figure S7 with corresponding key distances in SvS-A2 and SvS-WT given in Table S6.…”

Theoretical Study on the Mechanism of Spirocyclization in Spiroviolene Biosynthesis

“…In 2020, the stereochemistry of spiroviolene was revised by Snyder and colleagues on the basis of their total synthesis. 6) Then in 2021, Xu and Dickschat performed a mechanistic investigation of spiroviolene biosynthesis by means of stereoselective labeling experiments, and revised the biosynthetic pathway 7) (Fig. 1B).…”

Diterpenoids from Streptomyces: Structures, Biosyntheses and Bioactivities

“…Spiroviolene synthase, a type I DTS from S. violens NRRL ISP‐5597, was found to convert GGPP into spiroviolene ( 133 ) by in vitro enzyme assays, [61] and the formation mechanisms of 119 and 133 were further investigated by isotopic labelling experiments [61] . The stereochemical assignment of 133 has been revised based on a combination of total synthesis and investigations into the cyclization mechanism [64] . Characterization of spata‐13,17‐diene synthase (SpS), a type I DTS from S. xinghaiensis S187, demonstrated that it could convert GGPP into the main compound spata‐13,17‐diene ( 134 ), which has a 5–4–5 tricyclic skeleton, and the side compound prenylkelsoene ( 135 ), which has a 4–5–5 tricyclic skeleton [65] .…”