Towards a comprehensive understanding of the structural dynamics of a bacterial diterpene synthase during catalysis

Terpenes constitute the largest and structurally most diverse natural product family. Most terpenoids exhibit a stereochemically complex macrocyclic core, which is generated by C–C bond forming of aliphatic oligo-prenyl precursors. This reaction is catalysed by terpene synthases (TPSs), which are capable of chaperoning highly reactive carbocation intermediates through an enzyme-specific reaction. Due to the instability of carbocation intermediates, the proteins’ structural dynamics and enzyme:substrate interactions during TPS catalysis remain elusive. Here, we present the structure of the diterpene synthase CotB2, in complex with an in crystallo cyclised abrupt reaction product and a substrate-derived diphosphate. We captured additional snapshots of the reaction to gain an overview of CotB2’s catalytic mechanism. To enhance insights into catalysis, structural information is augmented with multiscale molecular dynamic simulations. Our data represent fundamental TPS structure dynamics during catalysis, which ultimately enable rational engineering towards tailored terpene macrocycles that are inaccessible by conventional chemical synthesis

Structural and Biochemical Investigations of Cyclases

Diterpenes belong to the versatile group of terpenes. These natural compounds consist of isoprene units and carry a wide variety of functions including anti-inflammatory and anti-viral properties. The precursor of diterpenes, geranylgeranyl diphosphate (GGPP), is cyclised by diterpene synthases, often referred to as diterpene cyclases and is then further chemically modified. Structural information is important to understand catalytic mechanism and specificity of diterpene cyclases. Thus far, only three crystal structures of diterpene cyclases are known, namely taxadiene synthase (TXS), abietadiene synthase and ent-copalyl diphosphate synthase, all of them deriving from plants. Here we present the structural determination of the bacterial diterpene cyclase cyclooctat-9-en-7-ol synthase (CotB2). CotB2 catalyses the cyclisation of GGPP to cyclooctat-9-en-7-ol, which is then further modified to the anti-inflammatory drug cyclooctatin. Recombinantly produced CotB2 yielded crystals that diffracted to 1.64 Å. The structure was solved via single-wavelength anomalous diffraction (SAD) with crystals of selenomethionine labelled CotB2. Interestingly, it is possible to alter the product of CotB2 by site directed mutagenesis in the active site as previously described by Görner et al.. Crystal structures of these mutants unveil the conformational changes compared to the wildtype and help answering the question of how a single point mutation leads to an alternative product. Apart from CotB2wt we also crystallised the mutant CotB2F149L

The first structure of a bacterial diterpene cyclase: CotB2

Sesquiterpenes and diterpenes are a diverse class of secondary metabolites that are predominantly derived from plants and some prokaryotes. The properties of these natural products encompass antitumor, antibiotic and even insecticidal activities. Therefore, they are interesting commercial targets for the chemical and pharmaceutical industries. Owing to their structural complexity, these compounds are more efficiently accessed by metabolic engineering of microbial systems than by chemical synthesis. This work presents the first crystal structure of a bacterial diterpene cyclase, CotB2 from the soil bacterium Streptomyces melanosporofaciens, at 1.64 Å resolution. CotB2 is a diterpene cyclase that catalyzes the cyclization of the linear geranylgeranyl diphosphate to the tricyclic cyclooctat-9-en-7-ol. The subsequent oxidation of cyclooctat-9-en-7-ol by two cytochrome P450 monooxygenases leads to bioactive cyclo­octatin. Plasticity residues that decorate the active site of CotB2 have been mutated, resulting in alternative monocyclic, dicyclic and tricyclic compounds that show bioactivity. These new compounds shed new light on diterpene cyclase reaction mechanisms. Furthermore, the product of mutant CotB2$^{W288G}$ produced the new antibiotic compound (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene, which acts specifically against multidrug-resistant Staphylococcus aureus. This opens a sustainable route for the industrial-scale production of this bioactive compound