Enzymatic control of product distribution in terpene synthases: insights from multiscale simulations

Raz, Keren; Levi, Shani; Gupta, Prashant Kumar; Major, Dan Thomas

doi:10.1016/j.copbio.2020.06.002

Cited by 32 publications

(55 citation statements)

References 103 publications

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“…The gas phase is a natural choice as a reference environment for terpene synthases, 16 as this reflects the inherent reactivity of the reacting species, although organic solvents may also be used. 16,56,59 The current gas-phase free energy profile is similar to that obtained by Sato et al 47 and Hong and Tantillo 46 and will only be briefly discussed. Similar to the work of Hong and Tantillo, we modeled the transformation A → I.…”

Section: ■ Resultssupporting

confidence: 75%

“…66,67 A combined quantum mechanics-molecular mechanics (QM/MM) potential was employed, in order to model the multistep cascade to form cyclooctat-9-en-7-ol (Scheme 1). 68−70 Similarly to our previous studies, 16,18 the QM region in CotB2 includes the substrate hydrocarbon framework, as well as the metal−pyrophosphate cluster PP−(Mg 2+ ) 3 , while the remaining enzyme−solvent system is represented by the CHARMM36 MM force field. 71,72 The QM region is described using the M06-2X functional, 52 with a mixed basis set (H, C, P: 6-31G(d); O: 6-31+G(d); Mg: STO-3G).…”

Section: ■ Methodsmentioning

confidence: 99%

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The Impression of a Nonexisting Catalytic Effect: The Role of CotB2 in Guiding the Complex Biosynthesis of Cyclooctat-9-en-7-ol

et al. 2020

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Terpene synthases generate terpenes employing diversified carbocation chemistry, including highly specific ring formations, proton and hydride transfers, and methyl as well as methylene migrations, followed by reaction quenching. In this enzyme family, the main catalytic challenge is not rate enhancement, but rather structural and reactive control of intrinsically unstable carbocations in order to guide the resulting product distribution.Here we employ multiscale modeling within classical and quantum dynamics frameworks to investigate the reaction mechanism in the diterpene synthase CotB2, commencing with the substrate geranyl geranyl diphosphate and terminating with the carbocation precursor to the final product cyclooctat-9en-7-ol. The 11-step in-enzyme carbocation cascade is compared with the same reaction in the absence of the enzyme. Remarkably, the free energy profiles in gas phase and in CotB2 are surprisingly similar. This similarity contrasts the multitude of strong π−cation, dipole−cation, and ion-pair interactions between all intermediates in the reaction cascade and the enzyme, suggesting a remarkable balance of interactions in CotB2. We ascribe this balance to the similar magnitude of the interactions between the carbocations along the reaction coordinate and the enzyme environment. The effect of CotB2 mutations is studied using multiscale mechanistic docking, machine learning, and X-ray crystallography, pointing the way for future terpene synthase design.

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Section: ■ Resultssupporting

confidence: 75%

Section: ■ Methodsmentioning

confidence: 99%

The Impression of a Nonexisting Catalytic Effect: The Role of CotB2 in Guiding the Complex Biosynthesis of Cyclooctat-9-en-7-ol

et al. 2020

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“…The product selectivity of (sesqui-) TPSs varies significantly within this versatile enzyme family ranging from single product formation (e.g., (+)-δ-cadinene synthase from Gossypium arboreum ) to a product portfolio of over 50 different compounds (e.g., γ-humulene synthase from Abies grandis ) [ 19 , 25 , 26 ]. In contrast to the common function of an enzyme as an accelerator of a reaction rate, the catalytic challenge for TPSs rather lies in the control of the highly reactive carbocation intermediates alongside their reaction trajectory [ 27 ]. The product distribution in TPSs is guided by several factors such as: (i) the activation of the C–O bond by the pyrophosphate-Mg 2+ -cluster in the active site, (ii) electrostatic interactions that lead to the sequestration of the active site, (iii) the specific positioning of water molecules or acidic/basic residues, that facilitate site-specific hydroxylations or (de)protonations, and (iv) a specific active site architecture that pre-shapes the carbocation intermediate [ 11 , 27 ].…”

Section: Resultsmentioning

confidence: 99%

“…In contrast to the common function of an enzyme as an accelerator of a reaction rate, the catalytic challenge for TPSs rather lies in the control of the highly reactive carbocation intermediates alongside their reaction trajectory [ 27 ]. The product distribution in TPSs is guided by several factors such as: (i) the activation of the C–O bond by the pyrophosphate-Mg 2+ -cluster in the active site, (ii) electrostatic interactions that lead to the sequestration of the active site, (iii) the specific positioning of water molecules or acidic/basic residues, that facilitate site-specific hydroxylations or (de)protonations, and (iv) a specific active site architecture that pre-shapes the carbocation intermediate [ 11 , 27 ]. For instance, for two fungal sesquiterpene synthases, Cop4 and Cop6 from Coprinus cinereus , it was demonstrated that a smaller carbocation binding pocket lead to a more specific product profile as the carbocation intermediate is more restricted along its potential cyclization routes [ 28 , 29 ].…”

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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“… How are the carbocation intermediates controlled in the catalytic pocket of sesterTPSs to produce different final products, thereby contributing to plant sesterterpene diversity? Computationally guided methods such as quantum mechanical/molecular mechanical molecular dynamics simulations have been employed to study the product distribution controlled by TPSs ( Sato et al., 2018 ; Raz et al., 2020 ). Together with the available AtTPS18 structure, multiscale simulations of sesterTPS will help us to engineer sesterTPS activity more rationally.…”

Section: Concluding Remarks and Perspectivesmentioning

confidence: 99%

Occurrence and biosynthesis of plant sesterterpenes (C25), a new addition to terpene diversity

Chen

et al. 2021

Plant Communications

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Enzymatic control of product distribution in terpene synthases: insights from multiscale simulations

Cited by 32 publications

References 103 publications

The Impression of a Nonexisting Catalytic Effect: The Role of CotB2 in Guiding the Complex Biosynthesis of Cyclooctat-9-en-7-ol

The Impression of a Nonexisting Catalytic Effect: The Role of CotB2 in Guiding the Complex Biosynthesis of Cyclooctat-9-en-7-ol

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

Occurrence and biosynthesis of plant sesterterpenes (C25), a new addition to terpene diversity

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