Catalytic diversity and homotropic allostery of two Cytochrome P450 monooxygenase like proteins from Trichoderma brevicompactum
Trichothecenes are the secondary metabolites produced by Trichoderma spp. Some of these molecules have been reported for their ability to stimulate plant growth by suppressing plant diseases and hence enabling Trichoderma spp. to be efficiently used as biocontrol agents in modern agriculture. Many of the proteins involved in the trichothecenes biosynthetic pathway in Trichoderma spp. are encoded by the genes present in the tri cluster. Tri4 protein catalyzes three consecutive oxygenation reaction steps during biosynthesis of isotrichodiol in the trichothecenes biosynthetic pathway, while tri11 protein catalyzes the C4 hydroxylation of 12, 13-epoxytrichothec-9-ene to produce trichodermol. In the present study, we have homology modelled the three-dimensional structures of tri4 and tri11 proteins. Furthermore, molecular dynamics simulations were carried out to elucidate the mechanism of their action. Both tri4 and tri11 encode for cytochrome P450 monooxygenase like proteins. These data also revealed effector-induced allosteric changes on substrate binding at an alternative binding site and showed potential homotropic negative cooperativity. These analyses also showed that their catalytic mechanism relies on protein–ligand and protein–heme interactions controlled by hydrophobic and hydrogen-bonding interactions which orient the complex in optimal conformation within the active sites.
KeywordsTrichoderma brevicompactum Cytochrome P450 monooxygenase Trichodiene 12, 13-epoxytrichothec-9-ene Homotropic cooperativity
Substrate recognition sites
Cytochrome P450 monooxygenases
Sequence analysis and data sets
The sequences for tri4 and tri11 proteins from Trichoderma brevicompactum were downloaded in FASTA format from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov.in). Secondary structure analysis was carried out using PSIPRED . The PSIPRED predicts the presence of α- helices, β-sheets and loops within a protein sequence. Multiple sequence alignment (MSA) was performed using MultAlin  between tri4, tri11, and cytochrome P450 s from other diverse organisms to find out conserved residues, motifs, and signature sequences. MultAlin uses an algorithm for the multiple alignments of sequences that is both accurate and precise. 3-D structures of ligands, TDN, and EPT were obtained from PubChem .
Homology modelling and structural analysis
Tertiary structure models for tri4 and tri11 proteins were generated by homology modelling using Phyre2 . The template used was a fungal cytochrome P450, lanosterol 14α-demethylase from yeast Saccharomyces cerevisiae (PDB ID; 4LXJ). Phyre2 predicted protein structure based on template 4LXJ with 100% confidence and 94% coverage for the query sequence of the tri4 protein and with 100% confidence and 98% coverage for the query sequence of the tri11 protein. The tri4 and tri11 protein sequences showed 37% and 44% overall homology with the template structure 4LXJ [25, 26, 27]. The modelled protein structures for both tri4 and tri11 proteins were energy minimized using Gromacs 4.6 package [9, 18, 19]; using the Gromos96 53a6 force field with a steepest descent optimization algorithm, protein immersed in water was subjected to energy minimization as reported earlier [17, 28, 29]. The system was immersed in a cubic box of simple point charge water molecules. Some of the Na+ counter-ions were added by replacing water molecules to keep charge neutrality of the simulated systems. The ligands were also energy minimized to obtain a stable structure. The 3-D models of proteins were validated by Ramachandran plot using RAMPAGE (http://mordred.bioc.cam.ac.uk/rapper/rampage.php) to assess the stereochemical quality of models.
Docking of TDN and EPT into the protein
TDN and EPT (ligands) were docked into the active sites of tri4 and tri11 proteins, respectively, to yield a binary complex using AutoDock Vina-2.0 software which is Monte Carlo-based. The ligands were docked at the conserved cysteine residue of CXG motif, reported for heme and substrate binding . The pdb files of proteins and ligands were converted to pdbqt files using AutoDock utility of MGLTools-1.5.6 [9, 21, 31] and the polar hydrogen atoms were added to all the receptor protein molecules. In AutoDock Vina, the coordinates of the predicted binding site residues were used to assign binding positions for the ligands. These pdbqt files of proteins and ligands were added to the conf.txt file which was used as input for AutoDock Vina. A grid was generated around the binding site of the dimensions 30, 30, and 30 in x-, y-, and z-directions, respectively. AutoDock Vina uses semi-empirical free energy force field to evaluate the ligand-binding conformation. To analyse allosteric behavior, another molecule of both the ligands was also docked at SRS-5 of tri4 and tri11 to yield a ternary complex.
Molecular dynamics simulations
Summary of tri4 and tri11 proteins simulation systems
tri4 protein (protein–heme)
tri4 protein–ligand binary complex
TDN in the active site
tri4 protein–ligand ternary complex
TDN in the SRS-5
tri11 protein (protein–heme)
tri11 protein ligand complex
EPT in the active site
tri11 protein–ligand ternary complex
EPT in the SRS-5
For analysis, the atom coordinates were recorded at every 10 ps during the simulation. Various parameters like root-mean-square deviations (RMSDs), root-mean-square fluctuations (RMSFs), hydrogen bonds, the distance between the center of masses of heme and ligands (TDN and EPT), and distances between interacting atoms of ligands and heme–iron were considered for analysis. Different time frames containing the reaction intermediates from simulation trajectories were analyzed using LigPlot+ and PyMOL. The hydrogen bonds mediated interactions and the hydrophobic contacts were analyzed using LigPlot+ and the distances were measured in PyMOL.
Sequence analysis of tri4 and tri11 proteins in T. brevicompactum showed conserved catalytic motifs and alternative SRSs
Interactions between proteins and ligands in the active site showed the amino acid residues involved in the catalysis
The ligands TDN and EPT were docked into an already reported active site that is present deep inside the proteins around the heme-binding region (Fig. 2c, d). The ligand TDN showed hydrophobic interactions with amino acid residues K110, T114, F118, A123, A125, A126, H133, V307, and A311 of tri4. The ligand EPT showed hydrophobic interactions with the active site residues R103, S107, I114, F115, T290, T293, A294, H480, and F481 of tri11 protein after docking. There were no hydrogen-bonding interactions between the proteins and the ligands observed after molecular docking.
Spatial arrangement of amino acid residues of heme-binding P450s like proteins to accommodate different reactants (TDN and EPT)
Summary of inter-molecular interactions of ligands (TDN and EPT) and heme with tri4 and tri11 encoded cytochrome P450 monooxygenase like proteins during simulations
Hydrogen-bond interactions between catalytic residues and heme propionates
Hydrogen-bond interactions between catalytic residues and TDN/EPT
Hydrophobic contacts between protein and TDN/EPT
K110, T127, V128, D129, H130, D131, H133, R137, R380, S381, R450, Q451
T114, F118, A123, A125, A126, R221, F144, V307, P308, A311, G312, T315, P377, R491
Y94, R103, D121, H122, R126, M364, N436, N441, V443, G444
R111, S107, A219, H480
T108, R111, I114, F115, F205, A218, A219, M222, T293, A294, S296, T298, V360, M364, H480, F481
The conformational flexibility of heme propionates is controlled by amino acid residues present in the catalytic site
Conserved cysteine residues were observed in the CXG motif of both the proteins that serve as a ligand to interact with heme–iron; in addition, in the case of tri4, sulfur atom of conserved cysteine residue was observed to be covalently linked with heme iron. During simulation, heme propionates showed hydrogen-bonding network with different amino acid residues, viz., K110, T127, V128, D129, H130, D131, H133, R137, R380, S381, R450, and Q451 of tri4 protein and Y94, R103, D121, H122, R126, M364, N436, N441, V443, and G444 of tri11 protein. Hydrophobic interactions of heme were also observed with different amino acid residues (Table 2). The hydrogen-bonding amino acid residues induced various conformations of heme propionates as observed during simulations. This conformational flexibility of heme propionates is mainly driven by interacting amino acid residues of catalytic site and is an important factor for P450 catalytic activity as reported earlier . Heme propionate interacted with arginine and asparagine repeatedly. The positive charge and amide group of asparagines could be the reason of these interactions. Since these residues have longer side chains, it may provide conformational flexibility for the interactions with heme propionates and help in accommodation of the substrates.
Changes in the active site of proteins caused by TDN and EPT binding
Helix-I present near heme exhibited catalytically important structural changes
Effect of allostery on P450 catalytic mechanism
Ligands were docked at the two binding sites reported earlier in the enzymes . Docking was carried out one at the catalytic site and another at the chosen allosteric site SRS-5. From the docking scores, it was demonstrated that at the SRS-5 site, the ligands showed more tight binding in comparison to the active site. The high binding affinity site SRS-5 is specific for substrate orientation in the active site; as reported earlier , the TDN and EPT were docked at this potential site of tri4 and tri11 proteins to yield a ternary complex to analyse potential allostery induced changes on the substrate catalysis.
The MD trajectory analyses of ternary complexes with two ligand molecules showed that high-affinity ligand-binding site SRS-5 functions as an allosteric site that may affect the catalysis at the active site ligand molecule. The EPT molecule bound at the active site showed a lower number of hydrogen bonds (with only amino acid residues H480 and R239) in the ternary complex in the presence of allosteric EPT molecule which has interacted with T362 at SRS-5. When the second molecule of TDN/EPT was docked at allosteric site SRS-5 of tri4 and tri11 in the ternary complex, the trajectory analyses showed that the relative distance between potential oxygenation atoms of TDN molecule bound at the active site and heme–iron was increased significantly, viz., C11, C13, and C2 at a distance of 5.8, 7.2, and 7.7 Å, respectively (Fig. 3b.3, c.3, a.3). Similarly, the trajectories analyses of tri11 ternary complex showed that the distance between C4 of active site EPT and heme–iron was increased to 6.6 Å from 5.6 Å (Fig. 4a.3). Two of the active site water molecules that were observed to show interactions with heme during MD trajectories analyses of binary complexes, disappeared in the ternary complexes, when the second ligand molecule was bound to allosteric site SRS-5 of both tri4 and tri11 proteins (Fig. 5c, d). The center of mass distances of ternary complexes indicated fewer fluctuations in active site ligand molecules for appropriate orientations for productive reaction. All these transitions of ternary complexes from binary complexes together pointed out at negative homotropic cooperativity which might be due to allosteric modulation of active site of protein by the incoming second ligand molecule located at a high binding affinity site SRS-5 (− 6.9 and − 7.6 kcal/mol) for tri4 and tri11 ternary complexes, respectively as reported earlier .
In this work, docking and MD simulations were performed to explore structural features that lead to the catalytic events occurring at the molecular levels in two cytochrome P450 monooxygenase like proteins (tri4 and tri11) from T. brevicompactum. These proteins showed common structural features and conserved sequence motifs that are important for the reaction. MSA of tri4 and tri11 proteins with other cytochrome P450s showed conserved amino acid residues. These conserved regions were observed to contain the catalytic site of the enzyme and other structurally important functional sequences. Our analysis showed that highly conserved motif FXXGXRXCXG is involved in heme-binding which contains the axial cysteine ligand of heme. This region contains the highly conserved cysteine residue which serves as fifth ligand residue in addition to the four coordination atoms from the porphyrin rings that coordinate with the heme iron and is located at position 452 in tri4 protein and at 442 in tri11 protein. The crystal structure of many P450s showed that this cysteine is absolutely conserved and is essential for P450 monooxygenase activity . The region around the heme possesses the highest conservation of amino acid residues as reported for other P450s, and most of these conserved residues contribute to the active site cavity. The motifs EXXR and PER are present in both tri4 and tri11 proteins, as reported earlier for E-R-R triads, the role of this triad has been reported in fungal P450s for locking the heme pocket in the active site cavity and stabilization of the active site core structure . Another motif AGXXT of cytochrome P450 monooxygenases is present in both tri4 and tri11 proteins which have been reported to be important for oxygen binding and activation. The C-terminal threonine residue of this motif lies in the active site cavity and may have a role in substrate binding during catalysis.
The cytochrome P450 monooxygenase catalytic cycle begins by binding of substrate in the active site in a favourable orientation with heme–iron, and the catalytic efficiency of the enzyme depends on substrate binding orientation and the distance of the potential atom of the substrate for oxygenation from the heme–iron. In the present study, the docking and MD simulation analysis on tri4 protein and protein–ligand complexes showed that after adopting the stability of system, the hydrophobic interactions of active site amino acid residues F118, A123, A125, A126, V307, F308, A311, P377, G312, and I491 with TDN in a binary complex positioned the substrate in a favourable orientation within the active site cavity with its potential atoms for hydroxylation and epoxidation reaction within a distance of 6 Å from the heme–iron (Fig. 3a.1, b.1, c.1), that is within an optimal distance for the reaction as reported earlier [23, 24]. The hydrophobic interactions with the ligand TDN are contributed by non-polar side chains of F118, A123, A125, A126, V307, F308, A311, P377, G312, and I491. These hydrophobic interactions may stabilize the substrate TDN in the binding site of tri4 encoded Cytochrome P450 monooxygenase like protein. The hydrophobic interactions of TDN with the polar side chain amino acid residues R221, R491, T315, and heme group could help in the reaction catalysis (Table 2). Arginine is a positively charged amino acid with guanidino group in the side chain, whereas threonine has a hydroxyl group and these amino acid residues may act as electrophiles as indicated earlier  and participate in proton transfer process that may lead to the reaction catalysis. We have not observed any hydrogen-bonding interactions between the substrate TDN and tri4 protein. Similarly, in tri11 protein, the docked protein–ligand complex showed that hydrogen-bonding interactions with various active site residues S107, R111, R239, A219, and H480, and EPT repositioned the ligand in a favourable orientation with its potential atoms for hydroxylation within a distance of 6 Å (Fig. 4a.2) from the heme–iron, which is chemically feasible distance for the reaction between C4 of EPT and heme–iron [33, 37]. S107 hydroxyl group interacted with the oxygen atom of EPT through hydrogen bonding; similarly, hydrogen bonding was also observed between guanidino group of R111 and the oxygen atom of EPT, between the amino group of A219 and the oxygen atom of EPT and between imidazole group of H480 and the oxygen atom of EPT. The side chains of these amino acid residues were observed to interact with their electrophilic atoms (N and O) with the ligand EPT. The electrophiles are involved in proton transfer process that leads to the oxygenation of the substrate. The changing hydrogen-bonding network could be correlated with the repositioning of ligand for better binding and the proton transfer process that ultimately lead to the catalysis . It was observed that the hydrophobic contacts by active site residues were established to block the entry of solvent which provided a hydrophobic environment for the reaction catalysis. Ionic interactions were also observed in the active site region of tri4 protein with heme propionate between positively and negatively charged amino acid residues, carboxylate group of D129 and the amino group of H133 and between D131 and H133, respectively, and ionic interaction between carboxylate group of D121 and amino group H122 of tri11 protein. These ionic interactions may provide stability to the cofactor heme and lock up the substrates TDN and EPT in the active site cavity. However, the ionic interactions between D121 and H122 of tri11 were lost in the ternary complex. Like substrate interacting residues, many active site residues were also observed to be forming hydrogen-bonding and hydrophobic interactions with the heme moiety in the binary complexes of both tri4 and tri11 (Table 2). More number of hydrogen bonds was observed between positively charged amino acid residues and heme propionates during the simulations; this could be correlated with the efficient process of proton transfer required for the reaction. This hydrogen-bonding network of active site residues with the heme propionates may play an important role in maintaining the catalytic activity of P450s as reported earlier for other cytochrome P450s [36, 42, 43]. The RMSDs of Cα atoms indicated that all the heme and substrate interacting residues were found to be stable during the simulation in both tri4 and tri11 proteins (Fig. S2).
The center of mass distances between ligands (TDN and EPT) and the heme–iron indicated that the ligands were located at a distance of 0.7 nm (Fig. S4a, b) in the active site cavity. This orientation of ligands may help them to interact with the heme–iron. However, in the case of tri4 protein, the ligand TDN again moved away from the heme–iron around 20 ns time of simulation. This fluctuation may be attributed to the little degree of motion in the active site and rearrangement of active site residues . The center of mass distances between ligands (TDN and EPT) and heme indicated that the movement of ligands and heme closer to each other within an ideal distance during simulation is associated with the stability of protein–ligand complexes and prepared the active site for productive catalysis.
The effect of allostery on P450 catalysis was also analyzed by docking a second ligand molecule in ternary complexes at allosteric site SRS-5 of tri4 and tri11 (Fig. 4b, c).This potential substrate binding site located around the EXXR motif has been reported for conserved residues that affect substrates orientation in the active site. The binding of second ligand molecule at this site may affect the proper positioning of ligand molecule in the active site as reported earlier, and similar results were obtained in the present study [21, 45]. The MD trajectories analyses of ternary complexes showed that the distance between the active site TDN/EPT and heme–iron has been increased significantly which may reduce the efficiency of the oxygenation reaction (Figs. 3a.3, b.3, c.3 and 4 a.3).The presence of second ligand molecule at SRS-5 and less hydrogen-bonding and hydrophobic interactions have limited the active site ligand molecule at an appropriate distance from heme–iron and the strong hydrophobic and hydrogen-bonding interactions were partially lost as compared to binary complexes . The active site EPT showed limited hydrogen-bonding interactions with only H480 and R239. The RMSDs and RMSFs analyses of ternary complexes showed more stability compared to the binary complexes. However, the RMSFs of tri4 ternary complex showed higher fluctuations than binary complex. This might be due to the nearest location of second TDN molecule to the active site (at a distance of 9 Å from heme–iron); this close proximity altered the active site more leading to an overall change in protein conformation, suggesting its allosteric behavior (Fig. S2c).The center of mass distances between the active site ligand molecules and heme–iron in ternary complexes during simulation indicated that ligands exhibited non-optimal orientations for a productive-binding site with their potential atoms moving away from the heme–iron (Fig. S4a, b).These results suggested that presence of second ligand molecules at allosteric site SRS-5 of tri4 and tri11 in ternary complexes leads to a conformational change at the active site of proteins that indicated the negative type of homotropic cooperativity.
Cytochrome P450 monooxygenases are important bio-catalysts involved in the substrate oxygenation and hydroxylation reaction steps during the biosynthesis of several fungal SMs. Our results on cytochrome P450 monooxygenase like proteins (tri4 and tri11) from T. brevicompactum which are involved in the trichothecenes biosynthetic pathway (Fig. 1) demonstrated that these two P450 like proteins may catalyze monooxygenation reactions mediated by heme cofactor and many of the inter-molecular interactions may lead to the catalysis. The MD analysis indicated that in tri4 and tri11 P450 like proteins, catalytic mechanism relies on the fact that direct protein–ligand and protein–heme interactions are promoted by hydrophobic and electrostatic forces with the efficiently controlled orientation of substrate within the active site and less structural and conformational changes in other parts of the proteins were observed. Our results also showed that homotropic negative cooperativity in ternary complexes of tri4 and tri11 proteins due to allosteric modulation of the active site in the presence of a second ligand molecule at alternative binding site, SRS-5 of both proteins may provide a way of feedback type of enzyme activity regulation. This work may be helpful to design in vitro experiments to elucidate further the catalytic mechanism of these two tri cluster genes encoded P450 like proteins involved in the production of agriculturally important SMs. This information may be helpful to the scientific workers in the field to better understand the catalytic mechanism of P450s which are involved at two crucial reaction steps of the biosynthetic pathway of trichothecene compounds. Therefore, this in silico study about their mechanisms may help to devise strategies to enhance/suppress the overall production of these molecules in biotechnological settings.
We acknowledge Central University of Himachal Pradesh and Bioinformatics Resources and Applications Facility, Centre for Development in Advanced Computing, Pune for providing the computational infrastructure. RH acknowledges National Fellowship for Higher Education from University Grants Commission, Govt. of India (UGC). SS receives research stipend from UGC. Research in YA lab is supported by extramural research funds from UGC, Science and Engineering Research Board (DST, Govt. of India), and Indian Council of Medical Research. We thank Dr. P. Aparoy for his generous help during the revision. Prof. Claudio Luchinat (editor-in-chief) and two anonymous referees are also sincerely acknowledged, whose insightful comments and advice during the editorial review helped us to improve our work enormously.
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