A Branched Diterpene Cascade: The Mechanism of Spinodiene Synthase from Saccharopolyspora spinosa

Abstract

Ad iterpene synthase from Saccharopolyspora spinosa was found to convert geranylgeranyl diphosphate into the new natural products spinodiene Aa nd B, accompanied by 2,7,18-dolabellatriene.T he structures and the formation mechanism of the enzyme products were investigated by extensive isotopic labelling experiments,w hich revealed an unusual branched isomerisation mechanism towards the neutral intermediate 2,7,18-dolabellatriene.ADiels-Alder reaction was used to convert the main diterpene product with its rare conjugated diene moiety into formal sesterterpene alcohols.

Mechanistic investigations on multiproduct β-himachalene synthase from Cryptosporangium arvum

“…A comparison of the 13 C NMR spectra from the incubations of HcS with (1 R )- and (1 S )-(1- 13 C,1- 2 H)FPP, resulting in a singlet for the ( R )-case and a triplet in the ( S )-case indicating a direct C–D bond, clearly demonstrated the stereospecific migration of H R from C-1. To complete the observations also for C-10, (2- 13 C)DMAPP was synthesised from (2- 13 C)-3-methylbut-2-en-1-ol [43] and incubated with (1 R )- or (1 S )-(1- 2 H)IPP [50], FPPS and HcS resulting in the expected opposite outcome than stated above, namely a triplet in the ( R )-case and a singlet for the ( S )-sample.…”

Diterpene Biosynthesis in Catenulispora acidiphila: On the Mechanism of Catenul‐14‐en‐6‐ol Synthase

“…The 1,3‐hydride shift from C1 to C11 was followed by using ( R )‐ and ( S )‐(1‐ 2 H)IPP, [18] which were used to elongate (7‐ 13 C)FPP with GGPPS to ( R )‐ and ( S )‐(1‐ 2 H,11‐ 13 C)GGPP, followed by cyclisation with CaCS (Figure S29). The hydride shift resulted in a direct 13 C− 2 H connection in 1 when using ( S )‐(1‐ 2 H)IPP, as indicated by an upfield shifted triplet peak (−0.47 ppm, 1 J C,D =19.0 Hz) for C11 in the 13 C NMR, while a singlet with a minor upfield shift (−0.01 ppm) was obtained with ( R )‐(1‐ 2 H)IPP, in agreement with a deuterium located two positions away.…”

Exploring novel bacterial terpene synthases

“…In a previous study, 262 presumptive bacterial TSs were identified and 27 proteins were functionally characterised in in vivo conditions [37]. Several other bacterial TSs have been characterised and their activities identified with single or multiple substrates [24,28,36,[38][39][40][41]. In this study, presumptive TS homologue protein sequences were clustered by pairwise similarity, and from the resulting neighbour-joining tree (Fig 1) we identified TSs from Gram-positive bacteria, mainly from the order Actinomycetales, as well as Gram-negative bacteria belonging to numerous orders.…”

“…This approach was first applied to bacterial TSs by Komatsu et al (2008) to identify 2-methylisoborneol synthases in Actinomycetes [25]. Using similar approaches, the number of uncharacterised bacterial terpene cyclases has recently increased to more than 600 regular class I terpene synthases, 400 geosmin synthases and over 120 2-methylisoborneol synthases, due to the ever-increasing availability of genome sequencing data [22,24,[26][27][28].…”