The archaeal highly thermostable GH35 family β‐galactosidase DaβGal has a unique seven domain protein fold

• Published in: The FEBS journal 2024-08, Vol.291 (16), p.3686-3705
• Main Authors: Kil, Yury, Pichkur, Evgeny B., Sergeev, Vladimir R., Zabrodskaya, Yana, Myasnikov, Alexander, Konevega, Andrey L., Shtam, Tatiana, Samygina, Valeriya R., Rychkov, Georgy N.
• Format: Article
• Language: English
• Subjects: Biotechnology; Eukaryotes; Fungi; Galactosidase; Glycosidases; Glycoside hydrolase; High temperature; hyperthermophilic Archaea; Lactose; lactose hydrolysis; Polypeptides; Protein folding; protein structure; β‐galactosidase
• ISSN: 1742-464X; 1742-4658; 1742-4658
• DOI: 10.1111/febs.17166

The most extensively studied β‐d‐galactosidases (EC3.2.1.23) belonging to four glycoside hydrolase (GH) families 1, 2, 35, and 42 are widely distributed among Bacteria, Archaea and Eukaryotes. Here, we report a novel GH35 family β‐galactosidase from the hyperthermophilic Thermoprotei archaeon Desulfurococcus amylolyticus (DaβGal). Unlike fungal monomeric six‐domain β‐galactosidases, the DaβGal enzyme is a dimer; it has an extra jelly roll domain D7 and three composite domains (D4, D5, and D6) that are formed by the distantly located polypeptide chain regions. The enzyme possesses a high specificity for β‐d‐galactopyranosides, and its distinguishing feature is the ability to cleave pNP‐β‐d‐fucopyranoside. DaβGal efficiently catalyzes the hydrolysis of lactose at high temperatures, remains stable and active at 65 °С, and retains activity at 95 °С with a half‐life time value equal to 73 min. These properties make archaeal DaβGal a more attractive candidate for biotechnology than the widely used fungal β‐galactosidases. We extended the list of the GH35 family β‐galactosidases with a new seven domain enzyme from the hyperthermophilic archaeon Desulfurococcus amylolyticus. The dimer structure of the DaβGal enzyme, obtained both by X‐ray analysis and Cryo‐EM, shares only certain structural similarities with fungal β‐galactosidases. It has an extra jelly roll domain D7 and three composite domains (D4, D5, and D6) that are formed by the distantly located polypeptide chain regions.