Mechanistic Characterization of the Fusicoccane-type Diterpene Synthase for Myrothec-15(17)-en-7-ol

Abstract

Fusicoccane (FC)-type diterpenes, featuring a common 5–8–5 tricyclic skeleton, possess diverse biological functions. Currently, only FC-type diterpene synthases (DTSs) for 3 of the 16 possible stereochemically distinct subtypes of the FC skeleton have been discovered. Herein, a (2si,6si,10re,14si)-FC-type DTS from Myrothecium gramineum is reported, which produces the diterpene alcohol myrothec-15(17)-en-7-ol. On the basis of homology modelings, the I189F variant was obtained by site-directed mutagenesis that prevents the penultimate carbocation from water attack, resulting in the deprotonation product myrotheca-7,15(17)-diene. Vibrational circular dichroism calculations in combination with chemical correlations through an enantioselective labeling strategy established the absolute configurations of these diterpenes. Density functional theory calculations revealed a water-mediated proton transfer mechanism, which is supported by isotope labeling experiments, in contrast to the deprotonation/reprotonation mechanism reported in previous studies.

1,2‐ or 1,3‐Hydride Shifts: What Controls Guaiane Biosynthesis?

“…For a similar step by the fungal myrothec‐15(17)‐en‐7‐ol synthase from Myrothecium gramineum (MgMS) recently a proton transfer mediated through two water molecules to induce further cyclization events was suggested based on DFT calculations (Scheme 5 A). [25] Such a mechanism could also be of interest for catalysis by PtTPS5 (Scheme 5 B). To investigate this hypothesis DFT calculations were started from protonated hedycaryol (Figure 2 ).…”

Two Diterpene Synthases from Chryseobacterium: Chryseodiene Synthase and Wanjudiene Synthase

“…Notably, 1 is structurally related to fusicoccane diterpenes (Scheme 2 A), including fusicocca-2,10(14)-diene (2) [16] that is made by the fusicoccadiene synthases from Phomopsis amygdali (PaFS) and Alternaria brassicicola (AbFS), [17] cyclooctat-9-en-7-ol (3) formed by CotB2 in Streptomyces melanosporofaciens, [18] cycloaraneosene (4) made by the DTS SdnA in Sordaria araneosa, [19] and the recently reported myrothec-15(17)-en-7-ol (5) produced by the DTS MgMS in Myrothecium graminearum. [20] Also the sesterterpene ophiobolin F (6), the product of AcOS in Aspergillus clavatus, [21] exhibits a highly similar core structure, but the cyclisation mechanisms of all these enzymes are different to the mechanism of CpCS, as a different stereochemistry is installed. To give an example, isotopic labelling experiments…”

Zwei Diterpensynthasen aus Chryseobacterium: Chryseodien‐Synthase und Wanjudien‐Synthase

“…Bemerkenswerterweise ist 1 strukturell verwandt mit den Fusicoccan‐Diterpenen (Schema A) wie Fusicocca‐2,10(14)‐dien ( 2 ), das durch die Fusicoccadien‐Synthasen aus Phomopsis amygdali (PaFS) und Alternaria brassicicola (AbFS) produziert wird, Cyclooctat‐9‐en‐7‐ol ( 3 ) gebildet durch CotB2 in Streptomyces melanosporofaciens , Cycloaraneosen ( 4 ) durch die DTS SdnA in Sordaria araneosa und dem jüngst berichteten Myrothec‐15(17)‐en‐7‐ol ( 5 ) produziert durch die DTS MgMS in Myrothecium graminearum . Auch das Sesterterpen Ophiobolin F ( 6 ), das Produkt von AcOS in Aspergillus clavatus , weist eine ähnliche Kernstruktur auf.…”