Structural studies of a cold-adapted dimeric β-D-galactosidase fromParacoccussp. 32d

Abstract

The crystal structure of a novel dimeric β-D-galactosidase from Paracoccus sp. 32d (ParβDG) was solved in space group P212121 at a resolution of 2.4 Å by molecular replacement with multiple models using the BALBES software. This enzyme belongs to glycoside hydrolase family 2 (GH2), similar to the tetrameric and hexameric β-D-galactosidases from Escherichia coli and Arthrobacter sp. C2-2, respectively. It is the second known structure of a cold-active GH2 β-galactosidase, and the first in the form of a functional dimer, which is also present in the asymmetric unit. Cold-adapted β-D-galactosidases have been the focus of extensive research owing to their utility in a variety of industrial technologies. One of their most appealing applications is in the hydrolysis of lactose, which not only results in the production of lactose-free dairy, but also eliminates the `sandy effect' and increases the sweetness of the product, thus enhancing its quality. The determined crystal structure represents the five-domain architecture of the enzyme, with its active site located in close vicinity to the dimer interface. To identify the amino-acid residues involved in the catalytic reaction and to obtain a better understanding of the mechanism of action of this atypical β-D-galactosidase, the crystal structure in complex with galactose (ParβDG-Gal) was also determined. The catalytic site of the enzyme is created by amino-acid residues from the central domain 3 and from domain 4 of an adjacent monomer. The crystal structure of this dimeric β-D-galactosidase reveals significant differences in comparison to other β-galactosidases. The largest difference is in the fifth domain, named Bgal_windup domain 5 in ParβDG, which contributes to stabilization of the functional dimer. The location of this domain 5, which is unique in size and structure, may be one of the factors responsible for the creation of a functional dimer and cold-adaptation of this enzyme.

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

“…Whereas the structures of multiple mesophilic βDGs are known, only two structures of cold-adapted βDGs have been previously deposited in the PDB [33,36] and the obtained results show that the investigated enzyme differs in tertiary and quaternary structure from the previously described ones. Here we describe the crystallization methods used to ameliorate and control crystallization of cold adapted ParβDG and ArthβDG, as well as crystal structure determination of ArthβDG, the second cold-active βDG from the GH2 family identified as dimeric up to a year ago.…”

“…The crystal structure of ParβDG has been already reported in our previous article [36] that focused on its structural analysis (PDB ID: 5EUV). The crystallization process was not discussed there in detail.…”

“…Since ParβDG purification has already been described [36], it will be mentioned only briefly. The protein was expressed in E. coli and purified using a two-step protocol employing ion exchange chromatography: the first step was carried out using Fractogel EMD DEAE column (Merck, Darmstadt, Germany) and was followed by a protein separation on a Resource Q column (Merck, Darmstadt, Germany).…”

“…The ArthβDG dimer interface is made by extensive contacts between Domain 1 interacting with Domain 5 of an adjacent monomer (Figure 7a). ParβDG acts also as a functional dimer, which is present in the asymmetric unit of the crystal [36]. The active site of these enzymes is created by residues belonging to both monomers of the dimer.…”

“…However, large GH2 βDGs were reported to be active as functional dimers, based on biochemical investigations [34,35]. The crystal structure of the first dimeric GH2 βDG was recently published by us [36]; however, that enzyme is much smaller than typical GH2 βDGs (a monomer of only 70 kDa) and exhibits a different shape and orientation of domain 5, called wind-up domain.…”