Model

Cinnamate 3-hydroxylase (C3H) is a crucial enzyme in the bioluminescent pathway, specifically in synthesizing caffeic acid, a key precursor of luciferin. By optimizing C3H and integrating it into the p-coumaric acid pathway via the TAL and PAL routes, we aimed to boost bioluminescence in plant systems. A deep understanding of how structural differences in C3H homologs affect bioluminescence can provide insights into the enzyme's performance, helping to amplify luciferin production and, consequently, the intensity of the bioluminescent output.

Our approach utilized an integrated computational strategy, including multiple sequence alignments (MSA), motif consensus analysis, secondary and tertiary structure prediction, root mean square deviation (RMSD) analysis, and docking site simulations. This enabled us to identify key structural features of C3H homologs and predict their influence on enzyme activity and bioluminescence enhancement.

Using BLAST, we identified eight homologous C3H proteins from six plant species, namely Arabidopsis thaliana (At), Catharanthus roseus (Cr), Nicotiana tabacum (Nt), Physcomitrium patens (Pp), Rosa chinensis (Rc), and Solanum lycopersicum (Sl). We aligned these sequences using Clustalw, producing an alignment file (.aln) to compare sequence homology.

The MSA (Fig. 1) visualization from SNAPgene revealed conserved residues across the eight C3H homologs. Conserved regions, marked in yellow, reflect critical functional motifs potentially influencing enzyme activity. Gaps in the alignment indicated sequence differences between the homologs, which may contribute to variations in enzyme efficiency.

Despite originating from diverse plant species, these homologs exhibit significant structural similarity, particularly in their functional motifs. The clusters of conserved amino acids suggest regions crucial for C3H function, likely involved in catalysis or structural integrity. However, the MSA alone is insufficient to explain functional differences, emphasizing the need for deeper structural analyses.

To identify conserved functional motifs, we employed the MEME suite for motif discovery. The analysis was constrained to a maximum of 15 motifs, with motif widths ranging from 6 to 100 amino acids. This allowed us to focus on the most critical and conserved regions.

Motif locations (Fig. 2) were largely conserved across all homologs. The motifs were distributed throughout the protein sequences, indicating that the homologs share core functional elements despite sequence variations in non-motif regions.

The alignment of motifs across the C3H homologs supports the hypothesis of conserved functional regions. These conserved motifs may correlate with enzyme efficiency in the bioluminescence pathway, though variations in non-motif regions could affect catalytic rates or substrate affinity. The insights gained here set the stage for structural analysis at the secondary and tertiary levels.

Using PSIPRED, we predicted the secondary structures of the eight C3H homologs, focusing on alpha helices, beta strands, and random coils.

The secondary structure prediction (Fig. 3) indicated that all homologs share similar secondary structural features. Alpha helices (pink) and beta strands (yellow) were consistent across the homologs, with variations in coiled regions (grey).

The strong correlation in secondary structure across the homologs suggests that overall folding patterns are conserved. This structural similarity hints at functional conservation, but small differences in coil regions may influence enzyme dynamics and substrate binding. These insights will be explored further through tertiary structure predictions and docking simulations.

We predicted the tertiary structures of the eight homologs using the SWISS-MODEL server, visualizing the structures with alpha helices in blue and beta sheets in green.

The tertiary structure models (Fig. 4) revealed high similarity among the homologs. Despite slight differences in surface loops and coiled regions, the overall folding pattern is conserved.

The tertiary structures align well with secondary structure predictions, confirming the structural conservation of C3H homologs. However, subtle variations in surface loops or coiled regions may contribute to functional differences, particularly in how the enzyme interacts with ligands like p-coumaric acid. These structural insights suggest that any enhancements in bioluminescence may result from small but crucial changes in these regions.

We performed RMSD analysis using TM-align to compare the tertiary structures of five C3H homologs. Arabidopsis thaliana (At) C3H served as the reference for comparison.

The RMSD values ranged from 1.18 to 2.15, with TM-scores between 0.87 and 0.97 (Fig. 5). These values indicate strong structural similarity, particularly between Physcomitrium patens (Pp) and At_CYP98A3, which exhibited the highest similarity (RMSD = 1.18).

RMSD analysis revealed significant structural similarity among the C3H homologs, with lower RMSD values indicating greater similarity to At_CYP98A3. This structural resemblance correlates with enhanced bioluminescence, as proteins with higher structural similarity to At_CYP98A3 tend to yield higher light intensity. These findings suggest that structural features of At_CYP98A3 may underlie its superior performance.

We simulated the docking of p-coumaric acid, the substrate for C3H, using AutoDock Vina. Ligand-binding sites were identified using ConSurf, focusing on functional and structural residues surrounding the active site.

The docking simulations (Fig. 7) revealed binding sites near conserved regions across the C3H homologs. Active sites for each protein homogy are calculated by Consurf.

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Key residues in the active sites, such as R436, P362, and G432 in At_CYP98A3, were identified as potential contributors to catalytic efficiency.

The docking simulations suggest that differences in active site composition and surrounding residues could explain variations in enzymatic performance. At_CYP98A3's superior performance in enhancing bioluminescence may stem from specific residue interactions within the active site that optimize the enzyme's catalytic activity. These findings align with the structural predictions and RMSD analysis, confirming that small structural variations can significantly impact bioluminescence efficiency.

To validate our computational predictions, we measured the light intensity in genetically modified tobacco plants expressing different C3H homologs. Light intensity was quantified using Image J, with the control being tobacco injected with only the water vector.

As shown in Fig. 6, tobacco expressing Pp_CYP98A3 exhibited the second-highest light intensity, closely following At_CYP98A3. This trend is consistent with the structural similarities observed in our RMSD analysis.

The empirical data support our hypothesis that structural similarity to At_CYP98A3 correlates with higher bioluminescence. Plants expressing homologs structurally closest to At_CYP98A3, such as Pp_CYP98A3, produced significantly brighter light. These findings highlight the importance of fine-tuning structural elements in C3H homologs to enhance bioluminescence output.

Our comprehensive analysis of C3H homologs, combining sequence alignment, structural predictions, and docking simulations, identified key structural features that contribute to enhanced bioluminescence. The similarity between At_CYP98A3 and other homologs, particularly Pp_CYP98A3, suggests that conserved structural motifs and active site residues are critical for optimizing enzyme performance. These insights will guide future engineering efforts to further enhance bioluminescence through targeted modifications of C3H homologs.