Identification of Pyruvate Dehydrogenase E1 as a Potential Target against Magnaporthe oryzae through Experimental and Theoretical Investigation

Abstract

Magnaporthe oryzae (M. oryzae) is a typical cause of rice blast in agricultural production. Isobavachalcone (IBC), an active ingredient of Psoralea corylifolia L. extract, is an effective fungicide against rice blast. To determine the mechanism of IBC against M. oryzae, the effect of IBC on the metabolic pathway of M. oryzae was explored by transcriptome profiling. In M. oryzae, the expression of pyruvate dehydrogenase E1 (PDHE1), part of the tricarboxylic acid (TCA cycle), was significantly decreased in response to treatment with IBC, which was verified by qPCR and testing of enzyme activity. To further elucidate the interactions between IBC and PDHE1, the 3D structure model of the PDHE1 from M. oryzae was established based on homology modeling. The model was utilized to analyze the molecular interactions through molecular docking and molecular dynamics simulation, revealing that IBC has π-π stacking interactions with residue TYR139 and undergoes hydrogen bonding with residue ASP217 of PDHE1. Additionally, the nonpolar residues PHE111, MET174, ILE 187, VAL188, and MET250 form strong hydrophobic interactions with IBC. The above results reveal that PDHE1 is a potential target for antifungal agents, which will be of great significance for guiding the design of new fungicides. This research clarified the mechanism of IBC against M. oryzae at the molecular level, which will underpin further studies of the inhibitory mechanism of flavonoids and the discovery of new targets. It also provides theoretical guidance for the field application of IBC.

Keywords: RNA sequencing; enzyme activity; homology modeling; isobavachalcone; molecular dynamics simulation; pyruvate dehydrogenase.

Figure 1

Pathway of the TCA cycle. The unigenes annotated by KEGG are marked with a green background. Blue and red borders mark DEGs.

Figure 2

Gene expression at different time points following treatment with IBC for (A) pyruvate dehydrogenase E1 component, (B) pyruvate dehydrogenase E2 component, (C) dihydrolipoamide dehydrogenase, (D) 2-oxoglutarate dehydrogenase E1 component, (E) 2-oxoglutarate dehydrogenase E2 component, (F) pyruvate carboxylase, (G) isocitrate dehydrogenase, and (H) isocitrate dehydrogenase (NAD+). The x-coordinate is the processing time, and the y-coordinate is the fold change normalized to the reference gene actin.

Figure 3

(A) Standard curves of 2,6-DCPIP. (B) The enzymatic activity of PDH within 16 h of treatment with isobavachalcone and DMSO.

Figure 4

(A) Chemical structure of isobavachalcone obtained by Marvin Sketch. (B) The schematic diagram of the template protein (PDB code: 3exe). (C) The final crystal structure of pyruvate dehydrogenase E1. It was constructed using chain A of G pyruvate dehydrogenase (E1p) component of the human pyruvate dehydrogenase complex as a template.

Figure 5

Validation of the homology model of pyruvate dehydrogenase. Ramachandran plots were obtained by Procheck on the website. The Ramachandran plot of the angles of phi and psi showed that the total number of residues was 346, with 92.8% of the residues in the most favorable region (271 residues).

Figure 6

Prediction of the theoretically active site of the pyruvate dehydrogenase E1 receptor. Highly conserved functional residues are enriched in regions 111 dynamics simulation, revealing that IBC has 121, 161–181, 211–231, 241–251 and 311–321.

Figure 7

Pivotal interaction site of isobavachalcone and the pyruvate dehydrogenase of M. oryzae.

Figure 8

(A) RMSD of Cα atoms, backbone atoms, side-chain atoms, and heavy atoms in PDH-IBC complexes over time, during 100 ns MD simulations. (B) RMSF of backbone atoms, Cα atoms, side-chain atoms, and heavy atoms of PHD-IBC complexes during 100 ns MD simulations. A green vertical line marks protein residues interacting with IBC.

Figure 9

(A) During molecular dynamics, the residues interacted with the ligand in the framework of each trajectory. The top panel shows the total number of specific contacts the protein made with the ligand throughout the trajectory. Some residues made more than one specific contact with the ligand, which is represented by a darker shade of orange, according to the scale to the right of the plot. (B) Protein-ligand interactions can be divided into four types: hydrogen bonding, hydrophobic, ionic, and water bridge. Each interaction type contains more specific subtypes, which can be explored through the “Simulation Interactions Diagram” panel. The stacked bar charts were normalized during the trajectory. Values over 1.0 are possible as some protein residues may make multiple contacts of the same subtype with the ligand.

Figure 10

MM-GBSA binding energy of pyruvate dehydrogenase-IBC complexes.