Multiscale Simulations on the Catalytic Plasticity of CYP76AH1

Qiu, Yufan; Diao, Hongjuan; Zheng, Ying; Wu, Ruibo

doi:10.3389/fchem.2021.689731

Cited by 6 publications

(6 citation statements)

References 54 publications

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“…Well-tempered metadynamics (WT-MTD) is a powerful enhanced sampling method that flattens the free energy surface to accelerate the occurrence of the rare events by periodically adding the time-dependent biasing potential on the specific collective variables (CVs), − and it has been widely used in the investigation of ligand binding or unbinding, drug-target interaction, and conformational dynamics. − In this study, 200 ns WT-MTD was employed to identify ligand-binding states with their free energy profiles, using NAMD 2.13 . The distance of substrate oxidation sites from the heme iron of P450 enzymes is often used to determine the metabolite type, and the distance should be less than 5 Å. ,,, Thus, the CVs were set to the distance between the two oxidation sites (C1′ and C4) and the heme iron in this work: Fe–C1′ and Fe–C4. The bin size is 0.1 Å, and the simulation temperature is 310 K. The Gaussian hill and width were set to 1 kcal/mol and 0.8 Å, respectively.…”

Section: Methodsmentioning

confidence: 99%

Molecular Insights into the Heterotropic Allosteric Mechanism in Cytochrome P450 3A4-Mediated Midazolam Metabolism

Zhang

Zheng

2022

J. Chem. Inf. Model.

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Cytochrome P450 3A4 (CYP3A4) is the main P450 enzyme for drug metabolism and drug–drug interactions (DDIs), as it is involved in the metabolic process of approximately 50% of drugs. A detailed mechanistic elucidation of DDIs mediated by CYP3A4 is commonly believed to be critical for drug optimization and rational use. Here, two typical probes, midazolam (MDZ, substrate) and testosterone (TST, allosteric effector), are used to investigate the molecular mechanism of CYP3A4-mediated heterotropic allosteric interactions, through conventional molecular dynamics (cMD) and well-tempered metadynamics (WT-MTD) simulations. Distance monitoring shows that TST can stably bind in two potential peripheral sites (Site 1 and Site 2) of CYP3A4. The binding of TST at these two sites can induce conformational changes in CYP3A4 flexible loops on the basis of conformational analysis, thereby promoting the transition of the MDZ binding mode and affecting the ratio of MDZ metabolites. According to the results of the residue interaction network, multiple allosteric communication pathways are identified that can provide vivid and applicable insights into the heterotropic allostery of TST on MDZ metabolism. Comparing the regulatory effects and the communication pathways, the allosteric effect caused by TST binding in Site 2 seems to be more pronounced than in Site 1. Our findings could provide a deeper understanding of CYP3A4-mediated heterotropic allostery at the atomic level and would be helpful for rational drug use as well as the design of new allosteric modulators.

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Section: Methodsmentioning

confidence: 99%

Molecular Insights into the Heterotropic Allosteric Mechanism in Cytochrome P450 3A4-Mediated Midazolam Metabolism

Zhang

Zheng

2022

J. Chem. Inf. Model.

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“…Classical and metadynamic MD simulations were employed to detect the differences in pocket kinetics of SmCY-P76AH1 D301E, V479F mutant and the wild type. [18] To examine the position and orientation of miltiradiene in CYP728B70, we established CYP728B70 structure prediction using RoseTTAFold protein modeling software, followed by MD simulations to obtain the binding conformation with miltiradiene. In the substrate-binding cavity of CYP728B70, miltiradiene is bound with six residues (Trp206, Val283, Ala284, Ile352, Phe353 and Pro457) through hydrophobic interaction.…”

Section: Substrate Docking and Site-directed Mutagenesis Of Cyp728b70mentioning

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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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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“…5.7 5.5 5.9 6.1 6.3 6.5 6.7 6.9 Double bond rearrangement in the C-ring of olefin 5 to yield stable aromatized 6 27 has been repeatedly reported to be a spontaneous reaction process 14,[28][29][30] . In our study, we transformed plasmids into engineered yeast BY-PS2 16 , which could self-produce 5 as a substrate.…”

Section: Cyp82d274 and Cyp82d263 Catalyze The Hydroxylation And Aroma...mentioning

confidence: 99%

Tandemly duplicated CYP82Ds catalyze 14-hydroxylation in triptolide biosynthesis and precursor production in Saccharomyces cerevisiae

Zhang

Gao

et al. 2023

Nat Commun

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Triptolide is a valuable multipotent antitumor diterpenoid in Tripterygium wilfordii, and its C-14 hydroxyl group is often selected for modification to enhance both the bioavailability and antitumor efficacy. However, the mechanism for 14-hydroxylation formation remains unknown. Here, we discover 133 kb of tandem duplicated CYP82Ds encoding 11 genes on chromosome 12 and characterize CYP82D274 and CYP82D263 as 14-hydroxylases that catalyze the metabolic grid in triptolide biosynthesis. The two CYP82Ds catalyze the aromatization of miltiradiene, which has been repeatedly reported to be a spontaneous process. In vivo assays and evaluations of the kinetic parameters of CYP82Ds indicate the most significant affinity to dehydroabietic acid among multiple intermediates. The precursor 14-hydroxy-dehydroabietic acid is successfully produced by engineered Saccharomyces cerevisiae. Our study provides genetic elements for further elucidation of the downstream biosynthetic pathways and heterologous production of triptolide and of the currently intractable biosynthesis of other 14-hydroxyl labdane-type secondary metabolites.

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Multiscale Simulations on the Catalytic Plasticity of CYP76AH1

Cited by 6 publications

References 54 publications

Molecular Insights into the Heterotropic Allosteric Mechanism in Cytochrome P450 3A4-Mediated Midazolam Metabolism

Molecular Insights into the Heterotropic Allosteric Mechanism in Cytochrome P450 3A4-Mediated Midazolam Metabolism

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

Tandemly duplicated CYP82Ds catalyze 14-hydroxylation in triptolide biosynthesis and precursor production in Saccharomyces cerevisiae

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