Mechanistic analysis for the origin of diverse diterpenes in Tripterygium wilfordii

Tu, Lichan; Cai, Xinbo; Zhang, Yifeng; Tong, Yuru; Wang, Jian; Su, Ping; Lü, Yun; Hu, Tianyuan; Luo, Yunfeng; Wu, Xiao-Ning; Li, Dan; Huang, Luqi; Gao, Wei

doi:10.1016/j.apsb.2022.02.013

Cited by 8 publications

(8 citation statements)

References 53 publications

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“…TMHMM‐2.0 (https://services.healthtech.dtu.dk/service.php?TMHMM‐2.0), InterPro‐92.0 (https://www.ebi.ac.uk/interpro/) and PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred) were used to predict transmembrane domains and protein secondary structures. The synonymous substitution rate ( K s) values and duplication time of CYP728B genes were calculated by K a K s Calculator 2.0 and the reported equation [23] …”

Section: Methodsmentioning

confidence: 99%

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Rational Design and Site‐Directed Mutagenesis Promote the Heterologous Production of the Antitumor Natural Product Dehydroabietic Acid

Ma,

Gao,

Qiu

et al. 2024

ChemistrySelect

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Cytochrome P450s participate in the biosynthesis of natural active ingredients and catalyze the formation of hydroxyl, carboxyl, epoxy and other groups from C−H bonds. CYP728B70 from Tripterygium wilfordii oxidizes the miltiradiene‐C18 methyl group to produce dehydroabietic acid, a diterpenoid intermediate with anticancer, antibacterial and anti‐inflammatory activities. To elevate the carboxylation product output, we excavated serveral homologous CYP728Bs and an N‐terminal truncated CYP728B70 transcript from T. wilfordii genome, of which CYP728B79 possessed a weak carboxylation effect towards miltiradiene through heterologous expression in Saccharomyces cerevisiae. We employed RoseTTAFold method and molecular dynamic simulations to predict its protein structure and docking pose of CYP728B70, and obtained several mutants with increased yield using Funclib and site‐directed mutagenesis methods. Our study confirmed the influence of peripheral residues on protein activity, improved the catalytic activity of CYP728B70, and would provide ideas for related enzyme modification.

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Section: Methodsmentioning

confidence: 99%

“…The synonymous substitution rate (Ks) values and duplication time of CYP728B genes were calculated by K a K s Calculator 2.0 and the reported equation. [23]…”

Section: Sequence Analysismentioning

confidence: 99%

Rational Design and Site‐Directed Mutagenesis Promote the Heterologous Production of the Antitumor Natural Product Dehydroabietic Acid

Ma,

Gao,

Qiu

et al. 2024

ChemistrySelect

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“…Except for arginine (R), cysteine (C), phenylalanine (F), and tryptophan (W), the effects of the other residues on enzyme function and product had been demonstrated by mutagenesis experiments. For example, the PdiTPSs OsKSL5i: I664T and OsKSL5i: I718V from rice (Oryza sativa) specifically produced ent-pimara-8( 14), 15-diene and ent-isokaur-15-ene, respectively (Xu et al, 2007); Six PdiTPSs from Tripterygium wilfordii independently evolved new functions by mutating specific residues, including TwKSL1v2: M607\T638A, TwKSL3: M608\I639, TwKSL2: A608\I639, TwCPS3: I115\N327\V328\H268, TwCPS5: T115\A327\T326, and TwCPS6: Y265 (Tu et al, 2022). Moreover, mutation of glutamic acid (E) at position 690 to arginine (R), phenylalanine (F), lysine (K), proline (P), or aspartic acid (D) or mutation of serine (S) at position 721 to valine (V) in SmMDS resulted in product loss (Tong et al, 2022), respectively.…”

Section: Residues Within 8 å Of Substrate Have More Impact On Productsmentioning

confidence: 99%

“…It had been also observed that some low-frequency residues, such as alanine (A) and histidine (H), contributed to product specificity. For example, the AgAS: A723S mutant of abietadiene synthase specifically produced pimaradienes, and the H268 residue in TwCPS3 also contributed to product specificity (Tu et al, 2022;Wilderman and Peters, 2007). Additionally, these low-frequency residues around the substrate often appeared in spatially conserved but physicochemically lowly conserved positions (Figs.…”

Section: Residues Within 8 å Of Substrate Have More Impact On Productsmentioning

confidence: 99%

“…The identified functional motifs include DXDD (Abbas et al, 2017), DDXXD (Rynkiewicz et al, 2001;Starks et al, 1997;Whittington et al, 2002), NSE\DTE (Degenhardt et al, 2009), PIX (Jia et al, 2017) and LHS...PNV (Potter et al, 2014;Potter et al, 2016a;Potter et al, 2016b). By examining the structure-function relationship of Selaginella moellendorffii miltiradiene synthase (SmMDS), specific residues around the substrate responsible for product specificity, such as E690, S717, and H721, were identified (Tong et al, 2022). Structural analysis and catalytic mechanism also suggest that the cavity formed by the substrate surrounding residues can selectively choose the substrate (Tao et al, 2022).…”

Section: Introductionmentioning

confidence: 99%

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Exploration of an enzyme-product mapping approach for plant-derived diterpene synthases

Zhao¹,

Liang²,

Han³

et al. 2023

Preprint

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Plant-derived diterpene synthases (PdiTPSs) play a critical role in the formation of structurally and functionally diverse diterpenoids. However, the relationship between PdiTPSs and the specificity or promiscuity of their products remains unclear. To explore this correlation, the sequences of 199 functionally characterized PdiTPSs and their corresponding 3D structures were collected and manually corrected. Using this compiled annotated database, the correlations among PdiTPSs sequences, domains, structures and their corresponding products were comprehensively analyzed. However, utilizing sequence similarity network (SSN), phylogenetic trees, and structural topology features alone was insufficient for effective functional classification of PdiTPSs as these methods could not establish a clear mapping between the enzymes and products. Surprisingly, residues verified to play a function through mutagenesis experiments were located within 8Å of the substrate. Aromatic residues surrounding the substrate exhibited selectivity towards its chemical structure. Specifically, tryptophan (W) was preferentially located around the linear substrate geranylgeranyl pyrophosphate (GGPP), while phenylalanine (F) and tyrosine (Y) were preferentially located around the initial cyclized diterpene intermediate. This analysis revealed the functional space of residues surrounding the substrate of PdiTPSs, most of which have not been experimentally explored. These findings provide guidance for screening specific residues for mutation studies to change the catalytic products of PdiTPSs, allowing us to better understand the correlation between PdiTPSs and their products.

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Engineering the microenvironment of P450s to enhance the production of diterpenoids in Saccharomyces cerevisiae

Cheng,

Luo,

Tang

et al. 2024

Acta Pharmaceutica Sinica B

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Mechanistic analysis for the origin of diverse diterpenes in Tripterygium wilfordii

Cited by 8 publications

References 53 publications

Rational Design and Site‐Directed Mutagenesis Promote the Heterologous Production of the Antitumor Natural Product Dehydroabietic Acid

Rational Design and Site‐Directed Mutagenesis Promote the Heterologous Production of the Antitumor Natural Product Dehydroabietic Acid

Exploration of an enzyme-product mapping approach for plant-derived diterpene synthases

Engineering the microenvironment of P450s to enhance the production of diterpenoids in Saccharomyces cerevisiae

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