Mechanistic investigations on six bacterial terpene cyclases

Abstract

SummaryThe products obtained by incubation of farnesyl diphosphate (FPP) with six purified bacterial terpene cyclases were characterised by one- and two-dimensional NMR spectroscopic methods, allowing for a full structure elucidation. The absolute configurations of four terpenes were determined based on their optical rotary powers. Incubation experiments with 13C-labelled isotopomers of FPP in buffers containing water or deuterium oxide allowed for detailed insights into the cyclisation mechanisms of the bacterial terpene cyclases.

Mechanistic Investigations of Two Bacterial Diterpene Cyclases: Spiroviolene Synthase and Tsukubadiene Synthase

“…Small‐scale reactions (1 mg) of the 20 isotopomers of ( 13 C 1 )GGPP with SvS followed by extraction with C 6 D 6 and direct 13 C NMR analysis without product purification showed the 13 C label in the expected positions in all cases (Figure S11). For the geminal methyl groups C16 and C17 of GGPP SvS showed a strictly controlled stereochemical course (Figures S11 R+S), as observed for most terpene cyclases ,. One of the few exceptions is (−)‐guaia‐6,10(14)‐diene synthase from Fusarium fujikuroi , which reveals a distribution of the label between C12 and C13 of FPP…”

Mechanisms of the Diterpene Cyclases β‐Pinacene Synthase from Dictyostelium discoideum and Hydropyrene Synthase from Streptomyces clavuligerus

“…One of the few enzymes causing a similar distribution of labelling is (1 R ,4 R ,5 S )‐guaia‐6,10(14)‐diene synthase (GdS) from Fusarium fujikuroi . Notably, the cyclisation reactions of GdS and PcS both proceed with a 1,2‐hydride shift into an i Pr group, while for systems with 1,3‐hydride shifts such as the TCs for T‐muurolol, 4‐ epi ‐cubebol and γ‐cadinene always a strict stereochemical course is found . These findings reflect the different topological constraints of the two possible reactions: For the 1,2‐hydride shift, the equal distances of the moving hydride to the two planes of the cation cause a scrambling of labelling in the i Pr group, while the well‐defined target plane for the 1,3‐hydride migration does not (Figure ).…”

Enzymatic Synthesis of Methylated Terpene Analogues Using the Plasticity of Bacterial Terpene Synthases

“…[15] In subsequent experiments bacterial terpene synthases were investigated for their potentialt oc onvert the methylated GPP and FPP analogues into methylated terpenes. For this purpose, an ewly identified linalool synthase from Chryseobacterium polytrichastri DSM 26 899 (CpLS, accession number WP 073293738) and the knownT -muurolols ynthase from Roseiflexus castenholzii DSM 13941 (TmS) [16] were selected, because these enzymes showed ap articularlye ffective conversion of their native substrates. While CpLS naturally accepts only GPP,b ut no FPP or GGPP,T mS shows most efficient conversion of FPPa nd also acceptsG PP,b ut no GGPP.T he main product obtained from GPP with purifiedC pLS ( Figure S19 Figures S20 andS 21).…”