Two Bacterial Diterpene Synthases from Allokutzneria albata Produce Bonnadiene, Phomopsene, and Allokutznerene

Abstract

Two diterpene synthases from Allokutzneria albata were studied for their products, resulting in the identification of the new compound bonnadiene from the first enzyme. Although phylogenetically unrelated to fungal phomopsene synthase, the second enzyme produced a mixture of phomopsene and a biosynthetically linked new compound, allokutznerene, as well as spiroviolene. Both enzymes were subjected to in-depth mechanistic studies involving isotopic labelling experiments, metal-cofactor variation, and site-directed mutagenesis. Oxidation products of phomopsene and allokutznerene are also discussed.

A Family of Related Fungal and Bacterial Di‐ and Sesterterpenes: Studies on Fusaterpenol and Variediene

“…Spirocyclic 11 is structurally similar to spirograterpene A ( 12 ) from Penicillium granulatum and to the bacterial compound spiroviolene ( 13 ) from Streptomyces violens , for which the diterpene synthase has recently been reported . Further recently characterised diterpene synthases include the cattleyene ( 14 ) synthase from Streptomyces cattleya and the allokutznerene ( 15 ) synthase from Allokutzneria albata , which produces a mixture of 15 and 1 . Furthermore, phomopsene synthases are known from Nocardia testacea and Nocardia rhamnosiphila that convert GGPP into 1 as a single product …”

“…The absolute configurations of 2 , 3 , 7 and 12 were determined by modified Mosher's method, and the absolute configuration of fungal 1 was deduced from that of 2 . The absolute configurations of bacterial 1 and the other bacterial diterpene synthase products 13 – 15 were determined by chemical correlation using enantioselectively deuterated GGPP isotopomers . For compound 4 , the crystalline sponge method was applied, an X‐ray‐based technique that allows structural data to be determined from small quantities of material .…”

Mechanistic Studies on Trichoacorenol Synthase from Amycolatopsis benzoatilytica

“…Compounds 3 and 4 separated from ent ‐ 3 and ent ‐ 4 of synthetic origin on a homochiral GC column (Figure S3), thus establishing the absolute configuration of the enzyme product as (−)‐ 1 , identical to previously described 1 from fungal sources. The same absolute configuration was independently concluded by comparing the NMR data of ent ‐ 1 (Table S2, Figures S4–S10) with HSQC spectra of labeled 1 obtained from enzyme incubations of the TS combined with FPP synthase from Streptomyces coelicolor and the selectively labeled samples (Table S3) ( R )‐ and ( S )‐(1‐ 13 C,1‐ 2 H)isopentenyl diphosphate (IPP) with IPP isomerase (IDI) from E. coli (Figure S11) or ( Z )‐ and ( E )‐(4‐ 13 C,4‐ 2 H)IPP elongating dimethylallyl diphosphate (DMAPP; Figure S12). The TS from A. benzoatilytica is therefore characterized as a (−)‐(4 R ,6 S ,7 S ,10 S )‐trichoacorenol ( 1 ) synthase (TaS).…”

“…Because TaS does not have similarity to any fungal TS, horizontal gene transfer, as discussed recently for corvol ether synthase genes from bacteria and fungi, is unlikely. Instead, a convergent evolution as for fungal and bacterial phomopsene synthases must be assumed. The cyclization mechanism of TaS was investigated in detail by isotopically labeled precursors and NMR analysis.…”

Diterpenbiosynthese in Actinomyceten: Studien an Cattleyensynthase und Phomopsensynthase

“…Die absolute Konfiguration von 1 wurde mit den stereoselektiv deuterierten Vorläufern ( E )‐ und ( Z )‐(4‐ 13 C,4‐ 2 H)IPP ermittelt, die mit DMAPP, der FPP‐Synthase aus S. coelicolor und GGPPS mit bekanntem stereochemischen Verlauf zu den korrespondierenden enantioselektiv deuterierten GGPP‐Isotopomeren umgesetzt und dann mit CyS zu markiertem 1 cyclisiert wurden (Schema A). Die deuterierten Kohlenstoffe fungieren als stereochemische Anker mit bekannter absoluter Konfiguration, die Rückschlüsse auf die absolute Konfiguration von 1 erlauben, indem die relative Orientierung der eingebauten Deuteriumatome ermittelt wird.…”