Azoreductases of <i>Pseudomonas</i> sp. PNPG3: a bioinformatics and molecular simulation study for azo dye detoxification

Synthetic dyes represent a significant environmental concern due to their persistence and toxicity. These compounds are extensively used across various industries, especially textiles, and are engineered to resist degradation, making them recalcitrant in natural ecosystems. This study focused on azoreductase (Azo) enzymes as potential bioremediation agents capable of degrading diverse azo dyes. Genome analysis of <i>Pseudomonas</i> sp. strain PNPG3 identified the presence of two distinct Azo genes, which were subsequently subjected to structural modelling. The models exhibited notably high aliphatic index values which are indicative of considerable thermostability. Structural validation of both models were performed using the ERRAT, PROCHECK, and QMEAN4 tools, confirming their reliability. Among the azo dyes evaluated, solvent black 3 exhibited the highest binding affinity, with a binding energy of -10.1 kcal/mol. It formed multiple h bonds and van der Waals interactions within the active sites of Azo-1. Congo red showed a maximum binding energy of -9.4 kcal/mol with Azo-2 from strain PNPG3, forming three h bonds within its active site. The Azo2-solvent black 3 complex exhibited the highest structural stability, as indicated by consistently low RMSD values averaging approximately 0.6 nm. Additionally, the complex maintained a low and uniform RMSF profile ranging from ∼0.2 to 0.34 nm, alongside the formation of multiple h bonds and a notable reduction in SASA, further suggested a more compact conformation with limited solvent exposure. PCA analysis further confirmed this stability. Overall, the study highlights the robust azo dye-degrading potential of strain PNPG3, emphasizing its promise for large-scale dye remediation.