The first structure of a bacterial diterpene cyclase: CotB2

Abstract

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.

Identification of amino acid networks governing catalysis in the closed complex of class I terpene synthases

“…diterpene synthases demonstrated a common H-bond network (SI Appendix, Fig. S1 D and E) (27,29,(33)(34)(35)(36)(37)(38)(39)(40)(41). This suggests that a conserved basic amino acid-rich triad sets and holds PP i in place during the complete catalytic cascade.…”

Multi-domain terpenoid cyclase architecture and prospects for proximity in bifunctional catalysis

“…While the simplest terpenoid cyclase structure is that of a single α domain class I enzyme, such as the bacterial sesquiterpene cyclase pentalenene synthase [10] or the diterpene cyclase CotB [24•], αβ, βγ, and αβγ domain architectures of increasing complexity are observed in both class I and class II plant diterpene cyclases (Figure 2); sometimes, αβγ cyclases are bifunctional enzymes in which tandem cyclization reactions are catalyzed by the class II and class I active sites. Intriguingly, αα domain architectures with (αα) n assemblies ( n = 1, 2, and 6) are also observed for terpenoid synthases, some of which catalyze tandem biosynthetic reactions.…”

An Unusual Terpene Cyclization Mechanism Involving a Carbon–Carbon Bond Rearrangement

“…Hypothetical mechanism originallyp roposed for the formation of cyclooctat-9-en-7-ol (2)f rom GGDP (1)catalyzed by CotB2. [2,4,5] OPP = pyrophosphate.…”