Abstract
The catalytic promiscuity and fidelity of cytochrome P450 enzymes are widespread in the skeletal modification of terpenoid natural products and have attracted much attention. CYP76AH1 is involved in key modification reactions in the biosynthetic pathway of tanshinone, a well-known medicinal norditerpenoid. In this work, classical molecular dynamic simulations, metadynamics, and DFT calculations were performed to investigate the protein conformational dynamics, ligand binding poses, and catalytic reaction mechanism in wide-type and mutant CYP76AH1. Our results not only reveal a plausible enzymatic mechanism for mutant CYP76AH1 leading to various products but also provide valuable guidance for rational protein engineering of the CYP76 family.
Highlights
Terpenoids, such as the well-known artemisinin, taxol, tanshinones, and ginsenosides, have received increasing attention for their multiple pharmaceutical values (Yang W. et al, 2020; Zheng et al, 2019)
The corresponding root-measure-square fluctuations (RMSFs) values of holo-(abietatrienebound) pocket residues in CYP76AH1D301E,V479F were much larger, whereas they became smaller in holo-state of wildtype CYP76AH1, suggesting that the active pocket becomes more flexible due to the ligand-induced fit effect and the D301E/V479F double mutant
The catalytic plasticity of the CYP76AH1 enzyme was investigated by multiscale simulations
Summary
Terpenoids, such as the well-known artemisinin, taxol, tanshinones, and ginsenosides, have received increasing attention for their multiple pharmaceutical values (Yang W. et al, 2020; Zheng et al, 2019). Principal component analysis (PCA), which recombines the original variables into a new set of unrelated variables to reflect principal information of original variables (Prompers and Bruschweiler, 2002), was performed to analyze the backbone atoms of pocket residues in wt/mut-abi-Rest systems. Pocket volumes of wt/mut-abi-Rest along the MD simulations were calculated by POVME3.0 (Wagner et al, 2017) and provided in Supplementary Figure S3A, and abietatriene was chosen to define the pocket with keyword DefinePocketByLigand, and the parameter GridSpacing was set to 1.0 Å. The representative structures of cluster analysis from MD simulation trajectory of systems wt-abi-Cpd I and mut-abi-Cpd I were used to generate cluster models (Supplementary Figure S4). The above theoretical methods and basis set size were used in previously computational studies of P450s (Dubey et al, 2016; Dubey et al, 2017; Su et al, 2019)
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