In Situ Random Microseeding and Streak Seeding Used for Growth of Crystals of Cold-Adapted beta-D-Galactosidases: Crystal Structure of betaDG from Arthrobacter sp. 32cB
There is an increasing demand for cold-adapted enzymes in a wide range of industrial branches. Nevertheless, structural information about them is still scarce. The knowledge of crystal structures is important to understand their mode of action and to design genetically engineered enzymes with enhanced activity. The most difficult task and the limiting step in structural studies of cold-adapted enzymes is their crystallization, which should provide well-diffracting monocrystals. Herein, we present a combination of well-established crystallization methods with new protocols based on crystal seeding that allowed us to obtain well-diffracting crystals of two cold-adapted beta-D-galactosidases (betaDGs) from Paracoccus sp. 32d (ParbetaDG) and from Arthrobacter sp. 32cB (ArthbetaDG). Structural studies of both betaDGs are important for designing efficient and inexpensive enzymatic tools for lactose removal and synthesis of galacto-oligosaccharides (GOS) and hetero-oligosaccharides (HOS), food additives proved to have a beneficial effect on the human immune system and intestinal flora. We also present the first crystal structure of ArthbetaDG (PDB ID: 6ETZ) determined at 1.9 Å resolution, and compare it to the ParbetaDG structure (PDB ID: 5EUV). In contrast to tetrameric lacZ betaDG and hexameric betaDG from Arthrobacter C2-2, both of these betaDGs are dimers, unusual for the GH2 family. Additionally, we discuss the various crystallization seeding protocols, which allowed us to obtain ParbetaDG and ArthbetaDG monocrystals suitable for diffraction experiments.
Maria Rutkiewicz-Krotewicz, Agnieszka Pietrzyk-Brzezińska, Marta Wanarska, Hubert Cieśliński, Anna Bujacz. (2018). In Situ Random Microseeding and Streak Seeding Used for Growth of Crystals of Cold-Adapted beta-D-Galactosidases: Crystal Structure of betaDG from Arthrobacter sp. 32cB, 8(1), 1-15. https://doi.org/10.3390/cryst8010013
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