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

Abstract

β-Galactosidase from Arthrobacter sp. 32cB (ArthβDG) is a cold-adapted enzyme able to catalyze hydrolysis of β-d-galactosides and transglycosylation reaction, where galactosyl moiety is being transferred onto an acceptor larger than a water molecule. Mutants of ArthβDG: D207A and E517Q were designed to determine the significance of specific residues and to enable formation of complexes with lactulose and sucrose and to shed light onto the structural basis of the transglycosylation reaction. The catalytic assays proved loss of function mutation E517 into glutamine and a significant drop of activity for mutation of D207 into alanine. Solving crystal structures of two new mutants, and new complex structures of previously presented mutant E441Q enables description of introduced changes within active site of enzyme and determining the importance of mutated residues for active site size and character. Furthermore, usage of mutants with diminished and abolished enzymatic activity enabled solving six complex structures with galactose, lactulose or sucrose bounds. As a result, not only the galactose binding sites were mapped on the enzyme’s surface but also the mode of lactulose, product of transglycosylation reaction, and binding within the enzyme’s active site were determined and the glucopyranose binding site in the distal of active site was discovered. The latter two especially show structural details of transglycosylation, providing valuable information that may be used for engineering of ArthβDG or other analogous galactosidases belonging to GH2 family.

β‐Galactosidase: Insights into source variability, genetic engineering, immobilisation and diverse applications in food, industry and medicine

“…For instance, de Maat et al (2019) found that deletion of the lacL gene, encoding the large subunit of the E. faecium b-galactosidase, caused the enzyme to be incapable of digesting the lactose, thus, decreasing the survival of E. faecium. Rutkiewicz et al (2020) found that mutation E517 into glutamine and D207 into alanine could lead to loss of function and a significant drop of activity for b-Galactosidase from Arthrobacter sp. 32cB.…”

“…Rutkiewicz et al . (2020) found that mutation E517 into glutamine and D207 into alanine could lead to loss of function and a significant drop of activity for β‐Galactosidase from Arthrobacter sp. 32cB.…”

Involvement of Versatile Bacteria Belonging to the Genus Arthrobacter in Milk and Dairy Products

“…C2-2, Arthrobacter psychrolactophilus strain F2, and other unclassified Arthrobacter spp. were found to produce β-galactosidases, which were active at an optimum temperature range of 0–50 °C [ 88 , 101 , 102 , 103 , 104 ]. As a result of these discoveries, cold-active β-galactosidases produced by A. psychrolactophilus strain F2 are likely suitable enzymes to use under low-temperature conditions due to their activity being highest at 0 °C while their optimum temperature and pH were 10 °C and 8.0, respectively [ 105 ].…”

Some Clues about Enzymes from Psychrophilic Microorganisms

“…It is worth noting that recently: (a) a hexameric β-galactosidase has been isolated from the psychrophilic Marinomonas ef1, and has shown remarkable stability against temperature, notwithstanding its highest activity at 5 • C [136]; (b) a dimeric cold-active β-galactosidase has been isolated from Arthrobacter sp. 32cB, and is considered of relevant interest for food industry [137,138].…”