Functional interpretation and structural insights of Arabidopsis lyrata cytochrome P450 CYP71A13 involved in auxin synthesis

Cytochrome P450 CYP71A13 of Arabidopsis lyrata is a heme protein involved in biosynthesis of indole-3-acetonitrile which leads to the formation of indolyl-3-acetic acid. It catalyzes a unique reaction: formation of a carbon-nitrogen triple bond and dehydration of indolyl-3-acetaldoxime. Homology model of this 57 kDa polypeptide revealed that the heme existed between H-helix and J- helix in the hydrophobic pocket, although both helixes are involved in catalytic activity, where Gly305 and Thr308, 311 of H- helix were involved in its stabilization. The substrate indole-3-acetaldoxime was tightly fitted into the substrate pocket with the aromatic ring being surrounded by amino acid residues creating a hydrophobic environment. The smaller size of the substrate binding pocket in cytochrome P450 CYP71A13 was due to the bulkiness of the two amino acid residues Phe182 and Trp315 pointing into the substrate binding cavity. The apparent role of the heme in cytochrome P450 CYP71A13 was to tether the substrate in the catalysis by indole-3-acetaldoxime dehydratase. Since the crystal structure of cytochrome P450 CYP71A13 has not yet been solved, the modeled structure revealed mechanism of substrate recognition and catalysis.


Homology modeling
The model building of cytochrome P450 CYP71A13 using human cytochrome P450 (PDB code 3QM4) was achieved using MODELLER 9.11 program [16, 17]. The model was built including the coordinates of heme group from 3QM4 during the modeling. The energy of the model was optimized by several rounds of energy minimizations with Swiss-PdbViewer [18], monitored manually to avoid major rearrangements of the structure. Bond lengths and angles were verified and optimized by consultation of Ramachandran maps followed by energy minimizations.

Figure 2:
Sequence alignment of human cytochrome P450 (P10635) and Arabidopsis lyrata cytochrome P450. The alignment was generated using the program MAFFT program (15). Red tubes denote α-helices and cyan β-sheets. Residues shown on a green background are involved in heme anchoring and residues on a magenta background are within five angstroms of the docked substrate.  : Overview of the modeled cytochrome P450 CYP71A13 structure. The overall structure of cytochrome P450 CYP71A13 was coloured blue to red from N-terminus to Cterminus. The letters indicate the name of the helices.

Substrate docking
Three dimensional structure of indolyl-3-acetaldoxime was prepared using ProDrg server [25] and docked in the model of cytochrome P450 CYP71A13 using the Autodock 4.1 program [26, 27] which uses a genetic algorithm as a global optimizer combined with energy minimization as a local search method. The active site was defined as a collection of amino acid residues enclosed within a sphere of 5 Å (1 Å=0.1 nm) radius centered on the catalytic triad of Arg84, His233 and Ser127. Autotors program of Autodock was used to define torsional degrees of freedom in the ligand. The minimization was achieved using Lamarckian genetic algorithm (LGA). The grid size was set to 40 x 46 x 40 points, centered on the heme in the crystal structure of the complex. Each docking run consisted of 100 independent docks with 1000 iteration cycles. A random start was used to generate the substrate position within the docking box. The substrate orientation giving the lowest interaction energy was chosen for additional rounds of docking.

Results & discussion:
The alignment used for building the homology model is shown in Figure 2 the amino acid sequence identity of cytochrome P450 CYP71A13 was 27% to 3QM4. Based on sequence alignment as an input, the MODELLER 9.11 software generated a large number of spatial restraints from the template structure and constructed a molecular model of cytochrome P450 CYP71A13. The possible applications of generated model depend mainly on the quality of the obtained model. The quality of generated model of cytochrome P450 CYP71A13 was evaluated by PROCHECK [28] which indicated that more than 98.5% of residues ɸ-ψ angles were in favoured and additional allowed region of Ramachandran plot ( Figure  3). Again it indicated that the finally obtained 3D model of cytochrome P450 CYP71A13 was satisfactory with respect to Ramachandran plot where only two residues were in disallowed region and four (1.0%) were in generously allowed regions. Residues located in unfavourable region were far from substrate binding domain indicating that these residues may not affect the ligand protein binding simulation. Analysis of physiochemical properties revealed that cytochrome P450 CYP71A13 had higher content of negatively and positively charged amino acids (62, 69 respectively) resulting in increased hydrophilic character Table 1

(see supplementary material).
The values of pI showed that plant cytochrome P450 CYP71A13 and cytochrome P450 CYP71E1 was stable at higher pH as compared to human cytochrome P450. The final models of cytochrome P450 CYP71A13 contained α+β structure consisting of ten major α-helices that form the conserved 3D fold of CYP proteins and seven β-sheets (Figure 4). The heme existed between H-helix and J-helix in the hydrophobic pocket where Gly305 and Thr308, 311 of H-helix were involved in its stabilization ( Figure 5D). Analysis of the surface of the structural model of cytochrome P450 CYP71A13 identified surface exposed hydrophilic domains in the region positioned near the N-terminal region while a hydrophobic core was present near the C-terminal region ( Figure 5A). The hydrophobic core was formed by Leu218, 370, 364, 452, Phe438, Pro446, Ile448 and Ala451 amino acids near the Cterminal region. This region seemed to be most likely the binding site for substrate.
Cytochrome P450 CYP71A13 converts indole-3-acetaldoxime to indole-3-acetonitrile. The substrate indole-3-acetaldoxime was docked into the substrate binding pocket so that the atoms involved in the initial dehydration reaction were positioned above the heme. The smaller size of the substrate binding pocket in cytochrome P450 CYP71A13 was due to the bulkiness of the two amino acid residues Phe182 and Trp315 pointing into the substrate binding cavity. Indole group of the oxime was positioned slightly closer to the heme iron and Oatom towards the Ser369 and His367 respectively with a final distance of 2.1Å and 4.3 Å (Figure 5B). The substrate indole-3acetaldoxime was tightly fitted into the substrate pocket with the aromatic ring being surrounded by residues creating a hydrophobic environment. Leu312, 370 and Pro368 from the Cterminal were positioned favourably for interaction with the indole group of the substrates in the proximal end of the pocket, while Ser369 in the heme distal end of the substrate pocket was positioned to interact with the leaving group of indole-3-acetaldoxime ( Figure 5C). The apparent role of the heme in cytochrome P450 CYP71A13 was to tether the substrate in the catalysis by indole-3-acetaldoxime dehydratase. This homology model revealed that hydrogen bonding between the OH group of indole-3-acetaldoxime and the side chains of Ser369 and His367 controls the specific orientation of the heme-bound substrate suitable for the elimination of the OH group of indole-3-acetaldoxime and that these residues and the heme created a prefixed site for substrate recognition and binding ( Figure 5C).

Figure 5:
Active site of cytochrome P450 CYP71A13 from Arabidopsis lyrata A) Surface representation of amino acid hydrophobicity in cytochrome P450 CYP71A13 with a color scale that varies from blue to white, representing dodger blue for the most hydrophilic, to white for the most hydrophobic residues; B and C show features of the substrate indole-3-acetaldoxime binding pocket of cytochrome P450 CYP71A13 and D represents amino acids involved in stabilization of heme in cytochrome P450 CYP71A