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

Dale L Ang,Saleem A Bokhari,Khawar Sohail Siddiqui,Gea Guerriero,Wallace J Bridge,Mubasher Zahir Hoque,Md Abir Hossain,Roberto Berni,Jean-Francois Hausman

doi:10.3390/molecules26030707

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

Highlights

Endochitinases (EC 3.2.1.14) randomly hydrolyze internal glycosidic linkages of insoluble chitin, which is a linear polymer of β-1,4-linked N-acetylglucosamine residues of soluble low molecular weight chitooligosaccharides (COSs) that are found in viruses, bacteria, archaea, plants, and animals, including mammals
M. marina (P-Mm; PDB, 4HMC) [10] and its mesophilic homolog from S. marcescens (MSm; PDB, 4AXN) [42], cold-adapted Arthrobacter Tad20 (ChiB-As; PDB, 1KFW) and its mesophilic homolog from S. proteamaculans (M-Sp; PDB, 4Q22), and hyperthermophilic chitinase from P. furiosus (T-Pf; PDB, 2DSK) [12] belonging to GH-18 family were obtained from the PDB (Protein Data Bank) and NCBI (National Center for Biotechnology Information) data banks
More and longer surface loops have been implicated in decreasing the stability of proteins, including chitinases

Summary

Introduction

Endochitinases (EC 3.2.1.14) randomly hydrolyze internal glycosidic linkages of insoluble chitin, which is a linear polymer of β-1,4-linked N-acetylglucosamine residues of soluble low molecular weight chitooligosaccharides (COSs) that are found in viruses, bacteria, archaea, plants, and animals, including mammals. Chitinases were suggested to be involved in the defense against fungi and other pathogens, as well as in inflammation [1]. Chitinases have been biotechnologically underexploited, but potential applications that are currently being explored [2,3,4] include fungicides and insecticides for use in agriculture, the bioconversion and bioremediation of chitin waste into single-cell protein, and value-added COSs and human health care treatments. A handful of thermally adapted chitinases from extremophilic organisms have been described [7,8,9], and currently, the X-ray structures of only two cold-adapted (Moritella marina [10,11]; Arthrobacter sp.) and a single thermophilic (Pyrococcus furiosus [12]) chitinases have been reported.

Methods

Discussion

Conclusion