Current understanding and biotechnological application of the bacterial diterpene synthase CotB2

Driller, Ronja; Garbe, Daniel; Mehlmer, Norbert; Fuchs, Monika; Raz, Keren; Major, Dan Thomas; Brück, Thomas; Loll, Bernhard

doi:10.3762/bjoc.15.228

Cited by 19 publications

(20 citation statements)

References 102 publications

(170 reference statements)

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“…The typical cyclopentane-type intermediate is always generated by cyclization between the cation (C1), the third alkenyl (C10C11), and the fourth alkenyl (C14C15) of the ionized polyprenyl diphosphate substrates, as shown in Scheme , to eventually afford the most widespread 5-X molecular skeletons products . Among them, cyclooctat-9-en-7-ol, the exclusive cyclization product of CotB2, serves as the key biosynthetic precursor of the promising anti-inflammatory drug candidate cyclooctatin. ,, Interestingly, the precise cyclization cascade of CotB2 can be reprogrammed through site-directed mutagenesis, leading to the generation of other bioactive diterpenoids, such as the dolabellane class with antibacterial activity and the cembranoid class with antineoplastic activity . Therefore, it is essential to comprehensively understand the CotB2-catalyzed reaction cascade, especially the precise control of chemoselectivity and stereoselectivity under native catalysis and the plastic activity of enzyme mutants.…”

Section: Introductionmentioning

confidence: 99%

Enzymatic Plasticity Inspired by the Diterpene Cyclase CotB2

et al. 2020

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Enzymatic plasticity, as a modern term referring to the functional conversion of an enzyme, is significant for enzymatic activity redesign. The bacterial diterpene cyclase CotB2 is a typical plastic enzyme by which its native form precisely conducts a chemical reaction while its mutants diversify the catalytic functions drastically. Many efforts have been made to disclose the mysteries of CotB2 enzyme catalysis. However, the catalytic details and regulatory mechanism toward the precise chemo- and stereoselectivity are still elusive. In this work, multiscale simulations are employed to illuminate the biocyclization mechanisms of the linear substrate into the final product cyclooctat-9-en-7-ol with a 5–8–5 fused ring scaffold, and the derailment products arising from the premature quenching of reactive carbocation intermediates are also discussed. The two major regulatory factors, local electrostatic stabilization effects from aromatic residues or polar residue in pocket and global features of active site including pocket-contour and pocket-hydrophobicity, are responsible for the enzymatic plasticity of CotB2. Further comparative studies of representative Euphorbiaceae and fungal diterpene cyclase (RcCS and PaFS) show a correlation between pocket plasticity and product diversity, which inspires a tentative enzyme product prediction and the rational diterpene cyclases’ reengineering in the future.

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Section: Introductionmentioning

confidence: 99%

Enzymatic Plasticity Inspired by the Diterpene Cyclase CotB2

et al. 2020

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“…S20) [ 19 , 26 , 29 ]. However, the impact of single amino acid substitutions in Copu5 and Copu9 is relatively small when compared to other TPSs (e.g., CotB2) [ 36 – 39 ]. In that context, residues lining the entrance of the active site (e.g., residues G186 and C187 in Copu5) affect product specificity.…”

Section: Resultsmentioning

confidence: 99%

Biotechnological potential and initial characterization of two novel sesquiterpene synthases from Basidiomycota Coniophora puteana for heterologous production of δ-cadinol

et al. 2022

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Background Terpene synthases are versatile catalysts in all domains of life, catalyzing the formation of an enormous variety of different terpenoid secondary metabolites. Due to their diverse bioactive properties, terpenoids are of great interest as innovative ingredients in pharmaceutical and cosmetic applications. Recent advances in genome sequencing have led to the discovery of numerous terpene synthases, in particular in Basidiomycota like the wood rotting fungus Coniophora puteana, which further enhances the scope for the manufacture of terpenes for industrial purposes. Results In this study we describe the identification of two novel (+)-δ-cadinol synthases from C. puteana, Copu5 and Copu9. The sesquiterpene (+)-δ-cadinol was previously shown to exhibit cytotoxic activity therefore having an application as possible, new, and sustainably sourced anti-tumor agent. In an Escherichia coli strain, optimized for sesquiterpene production, titers of 225 mg l−1 and 395 mg l−1, respectively, could be achieved. Remarkably, both enzymes share the same product profile thereby representing the first two terpene synthases from Basidiomycota with identical product profiles. We solved the crystal structure of Copu9 in its closed conformation, for the first time providing molecular details of sesquiterpene synthase from Basidiomycota. Based on the Copu9 structure, we conducted structure-based mutagenesis of amino acid residues lining the active site, thereby altering the product profile. Interestingly, the mutagenesis study also revealed that despite the conserved product profiles of Copu5 and Copu9 different conformational changes may accompany the catalytic cycle of the two enzymes. This observation suggests that the involvement of tertiary structure elements in the reaction mechanism(s) employed by terpene synthases may be more complex than commonly expected. Conclusion The presented product selectivity and titers of Copu5 and Copu9 may pave the way towards a sustainable, biotechnological production of the potentially new bioactive (+)-δ-cadinol. Furthermore, Copu5 and Copu9 may serve as model systems for further mechanistic studies of terpenoid catalysis. Graphical Abstract

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“…The diterpene synthase in fungi has been well studied to increase the production of diterpenoids or to synthesize new diterpenoids via heterologous expression ( Driller et al, 2019 ). However, there was no report about the function of diterpene synthase on the biosynthesis of triterpenes.…”

Section: Discussionmentioning

confidence: 99%

“…The identification of the sterols, which were biosynthesized from squalene, another polymer of IPP and DMAPP (Dhingra and Cramer, 2017), by GC-MS verified this conclusion. The diterpene synthase in fungi has been well studied to increase the production of diterpenoids or to synthesize new diterpenoids via heterologous expression (Driller et al, 2019). However, there was no report about the function of diterpene synthase on the biosynthesis of triterpenes.…”

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confidence: 99%

A Fungal Diterpene Synthase Is Responsible for Sterol Biosynthesis for Growth

et al. 2020

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A conserved open reading frame, dps , is described in Pestalotiopsis microspora , sharing a remarkable similarity with fungal diterpene synthases whose function is less studied. Loss-of-function approach manifested that dps was necessary for the growth and the development of the fungus. A deletion strain, dps Δ, showed a fundamental retardation in growth, which could deliberately be restored by the addition of exogenous sterols to the media. Gas chromatography–mass spectrometry analysis confirmed the loss of the ability to produce certain sterols. Thus, the tolerance and the resistance of dps Δ to several stress conditions were impaired. Secondary metabolites, such as the polyketide derivative dibenzodioxocinones, were significantly diminished. At the molecular level, the deletion of dps even affected the expression of genes in the mevalonate pathway. This report adds knowledge about fungal diterpene synthases in Pestalitiopsis microspora .

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Current understanding and biotechnological application of the bacterial diterpene synthase CotB2

Cited by 19 publications

References 102 publications

Enzymatic Plasticity Inspired by the Diterpene Cyclase CotB2

Enzymatic Plasticity Inspired by the Diterpene Cyclase CotB2

Biotechnological potential and initial characterization of two novel sesquiterpene synthases from Basidiomycota Coniophora puteana for heterologous production of δ-cadinol

A Fungal Diterpene Synthase Is Responsible for Sterol Biosynthesis for Growth

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