Structural studies of enzymes from thermophilic organisms and identification of their thermostability factors

Abstract

Thermophilic enzymes are characterized by their ability to resist temperature while maintaining their catalytic efficiency. They are mostly found in organisms, such as bacteria or fungi, that reside in natural habitats of high temperatures, usually between 50–80 °C. Thermophilic enzymes are of utmost importance for industrial and research applications and understanding their properties is key for their further improvement by genetic engineering approaches.

This study investigated four thermophilic enzymes, three from fungal sources and one from a bacterial source. X-ray crystallography technique was employed to extract the three-dimensional (3D) structural details of the enzymes. A brief description of the investigated thermophilic enzymes is given below. The crystal structure of a β-glucosidase from a thermophilic fungus (Chaetomium thermophilum), referred here as CtBGL, was determined to a resolution of 2.99 Å. CtBGL structure revealed the nucleophilic (Asp287) and acid/base (Glu517) catalytic residues. The structure of CtBGL showed a three- domain architecture as in other β-glucosidases but with variations in loops and linker regions. Glycosylation and charged residues were suggested as the potential thermostability contributing factors in CtBGL.

The crystal structure of an Auxiliary Activity 9 Lytic Polysaccharide Mono Oxygenase produced by the thermophilic fungus Thermoascus aurantiacus, referred to here as native TaAA9A (nTaAA9A), was determined to a resolution of 1.36 Å. nTaAA9A was found to be active in producing C1- and C4-oxidized products from cellulose and xylan. The nTaAA9A structure was compared with the recombinant form of the enzyme expressed in Aspergillus oryzae (rTaAA9A). The compared structures exhibited a root mean square deviation (RMSD) of 0.43 Å after structural superposition, suggesting subtle changes. Differences were observed in surface loops and glycosylation sites. nTaAA9A revealed higher degree of glycosylation than rTaAA9A. In nTaAA9A, Asn138 residue was found glycosylated with at least two NAG molecules. Glycosylation and electrostatic interactions were suggested as possible thermostability contributing factors.

The crystal structure of a Cu,Zn superoxide dismutase from a thermophilic fungus Chaetomium thermophilum (CtSOD) was determined to a resolution of 1.56 Å. CtSOD was crystallized with eight molecules (A-H) in the crystallographic asymmetric unit, resulting in eight distinct interfaces. Zn2+ was present in all subunits, but Cu2+ in 4 subunits only (C, D, E, and F). The active-site pocket region, along with the copper- and zinc-binding sites, were found highly conserved. A higher degree of oligomerization and an elevated contribution of polar residues in CtSOD were suggested as thermostability contributing factors.

The crystal structure of a carbonic anhydrase from a thermophilic bacterium Caloramator australicus (γ-CaCA) was determined to 1.11 Å resolution. This is the highest resolution thus far for a γ-family carbonic anhydrase. The enzyme was crystallized with 3 molecules in the asymmetric unit. The active site of each molecule was found at the interface of two neighbouring molecules. The γ-CaCA structure was found highly conserved, but differences were noticed in loop regions compared to other CAs. Charged residues and hydrophobic clusters were suggested as possible thermostability contributing factors in γ-CaCA.

Thermozymes hold a promising future for bio-economy and green chemistry. The results presented here could offer new ideas to develop sustainable and environment-friendly solutions for a better future.