Mechanistische Studien an zwei bakteriellen Diterpencyclasen: Spiroviolen‐Synthase und Tsukubadien‐Synthase

Abstract

Die Mechanismen von zwei Diterpencyclasen aus Streptomyceten – eine mit unbekanntem Produkt, das als der spirocyclische Kohlenwasserstoff Spiroviolen identifiziert wurde, und eine mit dem bekannten Produkt Tsukubadien – wurden detailliert durch Isotopenmarkierungsexperimente untersucht. Obwohl die Strukturen der Produkte sehr unterschiedlich sind, beginnen die Cyclisierungsmechanismen beider Enzyme mit derselben Cyclisierungsreaktion, bevor sie auf dem Weg zu ihren individuellen Produkten divergieren. Dies spiegelt die nahe phylogenetische Verwandtschaft der Enzyme wider.

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

“…The same experiment with (6‐ 13 C)FPP [20] gave singlets for C1 of 3 and 4 , again with an upfield shift for 4 , in line with deuterium incorporation at a neighboring position (Figure 1B), while (2‐ 13 C)FPP [20] in 2 H 2 O gave a singlet for C4 of 3 and an upfield shifted triplet for 4 , giving direct evidence for deuterium incorporation at C5 (Figure 1C), in line with path A and conflicting path B. The 1,3‐hydride shift in the biosynthesis of 3 and 4 was demonstrated by incubation of (2‐ 2 H)DMAPP [21] and (3‐ 13 C)IPP [22] with isopentenyl diphosphate isomerase from Escherichia coli [23] and FPP synthase (FPPS) from Streptomyces coelicolor [24] . This will yield a mixture of isotopomers of [3,7,11‐ 13 C 3 ,2,6,10‐ 2 H 3 ]FPP in which each terpene unit carries either a 13 C‐ or a 2 H‐labeling, but not both simultaneously.…”

Mechanistic Characterisation of Collinodiene Synthase, a Diterpene Synthase from Streptomyces collinus

“…The 1,2-hydride shift from D to E was also evident from an experiment with (2-2 H)FPP [33] and (3-13 C)IPP. [33] With this substrate combination the migrating deuterium ends up in a neighbouring position of the labelled carbon C-3, which influences its chemical shift in 1 and 6 (Δδ = À 0.11 ppm in both cases, Figure S62).…”

“…As a result of the hydride shift from C-2 to C-3 a direct 13 C-2 H bond is formed, evident from the upfield shifted triplets for C-3 of 1 (Δδ = À 0.49 ppm, 1 J C,D = 19.5 Hz) and of 6 (Δδ = À 0.47 ppm, 1 J C,D = 19.5 Hz). Along similar lines, the 1,2-hydride shift from D to E in the biosynthesis of 1 was investigated using (2-2 H)FPP [33] and (2-13 C)IPP [35] in combination with GGPPS and ScCotB2, resulting in the detection of an upfield shifted triplet for C-3 of 1 (Δδ = À 0.42 ppm, 1 J C,D = 19.2 Hz, Figure S62; the corresponding signal for 6 was too weak in this experiment). The 1,2-hydride shift from D to E was also evident from an experiment with (2-2 H)FPP [33] and (3-13 C)IPP.…”

“…Along similar lines, the 1,2-hydride shift from D to E in the biosynthesis of 1 was investigated using (2-2 H)FPP [33] and (2-13 C)IPP [35] in combination with GGPPS and ScCotB2, resulting in the detection of an upfield shifted triplet for C-3 of 1 (Δδ = À 0.42 ppm, 1 J C,D = 19.2 Hz, Figure S62; the corresponding signal for 6 was too weak in this experiment). The 1,2-hydride shift from D to E was also evident from an experiment with (2-2 H)FPP [33] and (3-13 C)IPP. [33] With this substrate combination the migrating deuterium ends up in a neighbouring position of the labelled carbon C-3, which influences its chemical shift in 1 and 6 (Δδ = À 0.11 ppm in both cases, Figure S62).…”

A Detailed View on Geosmin Biosynthesis

“…The enzymatic conversion of ( R )‐ and ( S )‐(1‐ 2 H)FPP [36] with AaGS and GC/MS analysis of the products revealed a specific loss of the 1‐ pro ‐ S hydrogen in 6 and 1 and a specific migration of the same hydrogen into the i Pr group of 10 (Figure S27), confirming earlier results with ScGS [28] . Furthermore, the usage of ( R )‐ and ( S )‐(1‐ 13 C,1‐ 2 H)FPP [37] established that the 1‐ pro ‐ R hydrogen remains bound to the original carbon in 1 and 6 , as indicated by a slightly upfield shifted triplet for C‐6 resulting from 13 C‐ 2 H spin coupling (Figure S28). Moreover, HSQC analysis of the product mixture obtained from ( R )‐(1‐ 13 C,1‐ 2 H)FPP demonstrated the α orientation of the deuterium atom in 1 (Figure S29).…”