Computational Analysis of Thermal Adaptation in Extremophilic Chitinases: The Achilles' Heel in Protein Structure and Industrial Utilization

Abstract

Understanding protein stability is critical for the application of enzymes in biotechnological processes. The structural basis for the stability of thermally adapted chitinases has not yet been examined. In this study, the amino acid sequences and X-ray structures of psychrophilic, mesophilic, and hyperthermophilic chitinases were analyzed using computational and molecular dynamics (MD) simulation methods. From the findings, the key features associated with higher stability in mesophilic and thermophilic chitinases were fewer and/or shorter loops, oligomerization, and less flexible surface regions. No consistent trends were observed between stability and amino acid composition, structural features, or electrostatic interactions. Instead, unique elements affecting stability were identified in different chitinases. Notably, hyperthermostable chitinase had a much shorter surface loop compared to psychrophilic and mesophilic homologs, implying that the extended floppy surface region in cold-adapted and mesophilic chitinases may have acted as a "weak link" from where unfolding was initiated. MD simulations confirmed that the prevalence and flexibility of the loops adjacent to the active site were greater in low-temperature-adapted chitinases and may have led to the occlusion of the active site at higher temperatures compared to their thermostable homologs. Following this, loop "hot spots" for stabilizing and destabilizing mutations were also identified. This information is not only useful for the elucidation of the structure-stability relationship, but will be crucial for designing and engineering chitinases to have enhanced thermoactivity and to withstand harsh industrial processing conditions.

Keywords: bioinformatics; biotechnology; chitinases; cold-adapted; enzyme; industrial applications; molecular dynamics (MD) simulations; protein structure–function–stability; thermophilic; thermostability.

Figure 1

Compositional features in thermally adapted chitinases. (a) The relative number of amino acid residues (ratio or %). (b) Gly and Pro in the secondary structures (%). All features were analyzed using the ProtParam tool or PDB DeepView. ↑, higher value of the parameter is associated with higher stability; ↓, higher value of the parameter is associated with lower stability. P, psychrophilic; M, mesophilic; T, thermophilic. Mm, M. marina; Sm, S. marcescens; As, Arthrobacter sp.; Sp, S. proteamaculans; Pf, P. furiosus.

Figure 2

Compositional and structural features in thermally adapted chitinases. (a) Secondary structure (SS) elements, accessible solvent area (ASA), types of amino acids, and total buried residues. (b) Various types of buried amino acids normalized to per 100 residues. SS elements were found using X-ray structures; total charged (DEHKR), hydrophobic (GAVLIPMFW), and uncharged polar (CNQSTY) components were found using ProtParam; average ASA and various types of buried residues were found using WHATIF. ↑, higher value of the parameter is associated with higher stability; ↓, higher value of the parameter is associated with lower stability. P, psychrophilic; M, mesophilic; T, thermophilic. Mm, M. marina; Sm, S. marcescens; As, Arthrobacter sp.; Sp, S. proteamaculans; Pf, P. furiosus.

Figure 3

Various interactions in thermally-adapted chitinases. (a) Total hydrophobic interactions and hydrogen bonds (H-bonds). (b) Normalized hydrophobic interactions and hydrogen bonds per 100 residues. (c) Total ionic and pi interactions. (d) Normalized ionic and pi interactions per 100 residues. Hydrophobic interactions (within 5 Å), H-bonds (dA:O, N, 3.5; S, 4.0 Å), ionic/salt bridges (within 4 Å), π-π (4.5–7.0 Å) and π-sulfur interactions (within 5.3 Å) were found using PIC; π-cation interactions (within 6 Å) were found using realistic electrostatics (CaPTURE) and the optimal H-bonding network was found using WHATIF. Higher values of interactions are associated with higher stability. P, psychrophilic; M, mesophilic; T, thermophilic. Mm, M. marina; Sm, S. marcescens; As, Arthrobacter sp.; Sp, S. proteamaculans; Pf, P. furiosus.

Figure 4

Backbone root-mean-square deviation (RMSD) and root-mean-square fluctuations (RMSF). (a,b) P-As and (c,d) T-Pf at 300 K (black) and 400 K (red). Identified primary floppy loops are highlighted in green with active sites in red.

Figure 5

RMS matrix histogram showing the abundance of the conformer ensembles at 400 K. P-As (black), T-Pf (blue), M-Sm (red), P-Mm (green), M-Sp (brown).

Figure 6

Multiple alignments of thermally adapted chitinases. Active-site residues (stars) within active sites (red) and loops (green). (*), Fully conserved residue; (:), conservation between groups of strongly similar properties; (.), conservation between groups of weakly similar properties.

Figure 7

Superimposition of the frames extracted from the 300 K (upper row) and 400 K (lower row) trajectories at 0 ns (brown), 100 ns (blue), 200 ns (purple), and 300 ns (green): (a) P-As, (b) T-Pf, (c) M-Sm, (d) P-Mm, and (e) M-Sp. Occlusion of the central cavity by adjacent loops was evident in the 400 K structures of P-As and P-Mm, and to a lesser extent in M-Sm and M-Sp, whereas T-Pf retained its structure. The RMS deviations at 400 K between 0 ns and 300 ns were 1.259, 1.021, 1.266, 1.207, and 1.133 Å, respectively.

Figure 8

Perspectives linked to the agricultural uses of improved chitinases. Research involving the modeling of extremozymes and rational design can lead to superior catalysts in terms of stability. Such enzymes can be expressed in heterologous hosts, purified, and used as biocontrol agents or to degrade chitinous waste to yield chitooligosaccharides (COSs). COSs can in turn be applied as elicitors to improve biotic stress responses in crops.