Mechanistic Characterisation of Bacterial Terpene Synthases from Chitinophagaceae Producing Marine-Type Diterpenes

Two neodolabellane-type diterpene synthases from Chitinophaga japonensis (CjCsS and CjNdtS) in addition to one enzyme from Dinghuibacter silviterrae (DsJS) were investigated. CjCsS produces the instable diterpene alcohol chitinosphaerol, which degrades upon chromatographic purification on silica gel to known presphaerol and isosphaerodienes I and II. Despite seemingly solid evidence in the literature, for all three compounds a structural revision is presented. Treatment of chitinosphaerol with N-bromosuccinimide resulted in the formation of the stable compounds bromosphaerols A and B. Phylogenetically unrelated CjNdtS catalyses the formation of neodolabella-1(14),2,7-triene, a compound that undergoes a remarkable thermal three-step sequence by Diels-Alder reaction to thermocyclene A, followed by a water-mediated rearrangement to thermocyclene B and double-bond isomerisation to thermocyclene C. Japonene A was identified as a side product, but is the main product of DsJS, where it is accompanied by japonene B and anaerol B. The good production of anaerol B by DsJS allowed reinvestigation of its recently addressed biosynthesis as a trace product of peyssonnosol B synthase AbPS2. All structure elucidations in this study were secured through stereoselective deuteration experiments, and special isotopic labelling experiments in conjunction with density functional theory (DFT) calculations revealed the cyclisation mechanisms of the investigated enzymes.