In Situ Random Microseeding and Streak Seeding Used for Growth of Crystals of Cold-Adapted β-d-Galactosidases: Crystal Structure of βDG from Arthrobacter sp. 32cB

Abstract

Abstract: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 β-D-galactosidases (βDGs) from Paracoccus sp. 32d (ParβDG) and from Arthrobacter sp. 32cB (ArthβDG). Structural studies of both βDGs 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 ArthβDG (PDB ID: 6ETZ) determined at 1.9 Å resolution, and compare it to the ParβDG structure (PDB ID: 5EUV). In contrast to tetrameric lacZ βDG and hexameric βDG from Arthrobacter C2-2, both of these βDGs are dimers, unusual for the GH2 family. Additionally, we discuss the various crystallization seeding protocols, which allowed us to obtain ParβDG and ArthβDG monocrystals suitable for diffraction experiments.

Active Site Architecture and Reaction Mechanism Determination of Cold Adapted β-d-galactosidase from Arthrobacter sp. 32cB

“…Cold-adapted Arth βDG possesses a functional dimer (not typical for the GH2 family). The dimer is stabilized by head-to-tail interactions between Domains 1 and Domains 5 from neighboring molecules [28]. The same oligomerization state, though shaped differently, was previously described by us for another cold-adapted β- d -galactosidase from Paracoccus sp.…”

Mapping the Transglycosylation Relevant Sites of Cold-Adapted β-d-Galactosidase from Arthrobacter sp. 32cB

“…β-d-Galactosidases belonging to the GH2 family are large proteins, usually~100 kDa, of which the active forms are: tetramers [2][3][4], hexamers [5], and as recently discovered dimers [6,7]. Their catalytic site can be divided into a galactose binding subsite and a platform for weak binding of different moieties [8].…”

“…32d [6], and further discussed here β-d-galactosidase from Arthrobacter sp. 32 cB [7]. Hence, detailed information on the structure of β-d-galactoside processing cold-adapted enzyme that could be used for prebiotics production at the industrial scale is still in demand.…”

“…Furthermore, it exhibits additional transglycosylation activity, it catalyzes synthesis of GOS and HOS, for example: lactulose and alkyl glycosides. [50] Architecture of its five domains [7], structural details of hydrolysis of lactose catalyzed by this enzyme [51], and its structural features responsible for the enzyme's cold adaptation [52] were investigated and discussed previously. Upon the solution of native structure, E441 and E517 were assigned as catalytic residues based on superposition of ArthβDG structure with lacZ E. coli β-d-galactosidase [7].…”

“…[50] Architecture of its five domains [7], structural details of hydrolysis of lactose catalyzed by this enzyme [51], and its structural features responsible for the enzyme's cold adaptation [52] were investigated and discussed previously. Upon the solution of native structure, E441 and E517 were assigned as catalytic residues based on superposition of ArthβDG structure with lacZ E. coli β-d-galactosidase [7]. The role of residue E441 was previously proven: a loss-of-function mutant E441Q was designed, a biochemical activity assay was performed, and the protein was crystallized.…”