Chitosan, a polysaccharide derived from chitin, is gaining importance due to its biocompatibility and potential applications in various fields after degradation to chitosan oligosaccharides. Enzymatic degradation using chitosanase offers an environmentally friendly alternative to chemical and physical methods. Chitosanases, produced by bacteria and fungi, are categorized into families based on their structure and function (GH5, 7, 8, 46, 75, 80, and 2, 35). The study focuses on chitosanase from Aspergillus fumigatus Y2K, previously shown to degrade chitosan but exhibiting poor thermal stability above 55°C despite an optimal temperature of 65-70°C. The enzyme belongs to the GH75 family, with Asp143 and Glu152 identified as catalytic residues. This research hypothesizes that high temperatures induce structural changes hindering ligand binding and reducing activity stability. To investigate this, the researchers use Alphafold2 to predict the structure, validated by multiple evaluation tools and compared with existing experimental structures. Molecular dynamics (MD) simulations at different temperatures (300K and 350K) were employed to analyze structural and dynamic changes and their impact on thermal stability. The combination of structure prediction and dynamic simulations provided detailed insights into the factors determining the chitosanase's thermal stability.

The literature review extensively covers previous research on chitosanases, their classification within glycosyl hydrolase families, and their applications. It highlights the optimal reaction temperatures and pH for various microbial chitosanases, emphasizing the common challenge of poor thermal stability at higher temperatures, limiting industrial applications. The review specifically addresses the existing knowledge about chitosanase from Aspergillus fumigatus, including its molecular weight, optimal reaction conditions, and identified catalytic residues (Asp160 and Glu169, or Asp143 and Glu152 after signal peptide removal), based on previous studies. The lack of sufficient experimental data on GH75 chitosanase structures prompted the researchers to leverage Alphafold2 for structure prediction and validation.

The study employed a multifaceted computational approach. First, the researchers used Alphafold2 to predict the 3D structure of the chitosanase from Aspergillus fumigatus (Q875I9, with signal peptide removed). The accuracy of Alphafold2 was initially validated by comparing its predictions for four chitosanases with known experimental structures (GH2, GH8, GH46, and GH80). The predicted structure of Q875I9 was thoroughly evaluated using PROCHECK, VERIFY_3D, and PROSA to ensure its reliability. The predicted structure was analyzed to identify secondary structural elements (α-helices, β-sheets, and loops) and the binding site using DeepSite. Protonation states of residues were determined using H++ at pH 6.5. Molecular docking of DP6-chitosan (a chitosan oligomer) to the predicted binding site was performed using Autodock Vina to determine the binding mode. Molecular dynamics (MD) simulations were conducted using AMBER-tools22 and Gromacs2022.1 at two temperatures, 300K and 350K. Three independent 100 ns simulations were performed for the enzyme-only system at each temperature, and three 50 ns simulations were performed for the enzyme-DP6 complex at 300K. Various analyses were applied to the simulation trajectories, including RMSD, RMSF, radius of gyration, dynamic cross-correlation, PCA, cluster analysis, and hydrogen bond and salt bridge analysis. Finally, MMPBSA was used to calculate the binding free energy and its decomposition into per-residue contributions to understand the influence of residue flexibility on binding.

Alphafold2 accurately predicted the chitosanase structure, exhibiting high similarity to the experimentally determined structure of V-CSN. The binding site was identified, revealing its interaction with DP6-chitosan. MD simulations showed increased flexibility in the binding site at 350K compared to 300K. Specifically, loop6 exhibited a significant downward shift at 350K, closing the binding site and hindering substrate binding. The analysis of hydrogen bonds revealed a sharp decrease in the time proportion of important hydrogen bonds at the binding site at high temperature, suggesting disruption of hydrogen bonds as the main factor for conformational changes. Importantly, residues energetically contributing to chitosan binding were located within the highly flexible regions, leading to the observed decreased activity stability. Dynamic cross-correlation analysis showed that the correlated motion among regions forming the binding site was reduced at high temperature. PCA and cluster analysis identified a dominant conformation at 350K with a closed binding site, confirming the impact of high temperature on binding. Analysis of hydrogen bonds and salt bridges revealed that hydrogen bond disruption within the highly flexible region was the primary cause of conformational change. Conversely, salt bridges showed good stability. The removal of disulfide bonds from the model significantly decreased stability at both temperatures, particularly at 350K. Finally, MMPBSA analysis indicated that residues with the lowest binding energies were located in the highly flexible region of the binding site; their high flexibility negatively affected binding capacity.

The findings directly address the research question by demonstrating that the reduced thermal stability of the Aspergillus fumigatus chitosanase is due to the increased flexibility of the binding site at high temperatures, leading to a conformational change that hinders substrate binding. The high similarity between the predicted structure and V-CSN validates the accuracy of the Alphafold2 prediction and supports the use of computational methods for studying proteins without experimental structures. The significant role of hydrogen bonds in maintaining binding site stability is highlighted, providing targets for future protein engineering efforts aimed at improving thermal stability. The observed interplay between flexibility, hydrogen bond disruption, and binding free energy contributes significantly to the understanding of protein thermal stability and the impact of local structural changes on overall function.

This study successfully predicted the structure of Aspergillus fumigatus chitosanase using Alphafold2 and characterized its dynamic behavior under different temperatures. The findings elucidate the structural and dynamic basis for its thermal instability, specifically pointing to the high flexibility of the binding site and the disruption of hydrogen bonds as key factors. The identified highly flexible region offers promising targets for site-directed mutagenesis to improve the thermal stability of this enzyme. The computational approach demonstrated here underscores the potential of using structure prediction and MD simulations to study proteins lacking experimental structures, opening up new avenues in protein engineering and biotechnology.

The study relies entirely on computational methods, and experimental validation of the predicted structure and dynamic behavior is necessary to fully confirm the findings. The simulations, while extensive, may not capture all possible conformations and dynamics of the protein. The accuracy of the force field used in the simulations also influences the results. Furthermore, the study focuses on a specific chitosan oligomer (DP6) and does not fully address the enzyme's interactions with other chitosan chain lengths.