PCPD: Plant cytochrome P450 database and web-based tools for structural construction and ligand docking

Abstract

Plant cytochrome P450s play key roles in the diversification and functional modification of plant natural products. Although over 200,000 plant P450 gene sequences have been recorded, only seven crystalized P450 genes severely hampered the functional characterization, gene mining and engineering of important P450s. Here, we combined Rosetta homologous modeling and MD-based refinement to construct a high-resolution P450 structure prediction process (PCPCM), which was applied to 181 plant P450s with identified functions. Furthermore, we constructed a ligand docking process (PCPLD) that can be applied for plant P450s virtual screening. 10 examples of virtual screening indicated the process can reduce about 80% screening space for next experimental verification. Finally, we constructed a plant P450 database (PCPD: http://p450.biodesign.ac.cn/), which includes the sequences, structures and functions of the 181 plant P450s, and a web service based on PCPCM and PCPLD. Our study not only developed methods for the P450-specific structure analysis, but also introduced a universal approach that can assist the mining and functional analysis of P450 enzymes.

Keywords: Database; Homologous modeling; Ligand docking; Plant P450 database; Web service.

Fig. 1

Data of plant cytochrome P450 enzymes with known functions. (A) The phylogenetic tree of 181 plant P450s with known functions. The outermost ring indicated the types of substrates, and the internal ring indicated the types of catalytic reactions. (B) Structural alignment of five plant P450 protein crystals in PDB. The pairwise amino acid identities were shown on the structures.

Fig. 2

PCPCM workflow and model evaluations. (A) The workflow of PCPCM. The workflow mainly includes: preprocessing of the input sequence (TMHMM step) and blast search (BLAST+ step); homology modeling to predict initial model (RosettaCM step); addition of active heme prosthetic group (Add HEM step); molecular dynamics optimization of the composite structure (Amber step). (B) Comparison of five structure prediction methods for P450 structural modeling. For each crystal structure, five methods were ranked based on the protein structure quality measures. Ranking first represents the method that had the best performance for the corresponding crystal structure and quality measure. Each color represents a modeling method, the Y-axis represents the number of five rankings of each method. (C) Comparison of RMSD values of five structure prediction methods. The RMSD values were calculated by comparing the crystal structures with the modeling structures from five methods using LGA (http://proteinmodel.org/AS2TS/LGA/lga.html).

Fig. 3

Docking workflow and evaluations of PCPLD. (A) The Docking workflow of PCPLD. (B) Plant P450 crystal structures (gray) and predicted structures (purple) docking with corresponding ligands, plant P450 crystal structure (PDB ID: 3DSK, 6A15, and 6J95), and corresponding ligands (vanillic acid, cholesterol, and retina). Since there are no corresponding ligands and analogs in the crystal structure of 5YLW and 6B9Y, there is no docking. (C) 10 ligands and 181 plant P450s were selected for cross-docking, and the results were shown in the figures. The red marked short-term represented the correct docking. The 10 ligands are abbreviated as Te (terpenoids), Fa (fatty acids), Aa (amino acids), Fl (flavonoids), and Al (alkaloids).

Fig. 4

The main function interface of PCPD. (A) The data browsing and downloading interface of the website. It mainly includes: browsing of plant P450 family, catalytic and function and reaction, as well as downloading of sequence and structure. (B) The application interface of PCPCM and PCPLD. Users can choose the needed method and submit input file according to the User Guide.