bi7b00207_si_001.pdf (306.22 kB)
Biochemical Characterization of the Functional Roles of Residues in the Active Site of the β‑Galactosidase from Bacillus circulans ATCC 31382
journal contribution
posted on 2017-05-24, 00:00 authored by Huifang Yin, Tjaard Pijning, Xiangfeng Meng, Lubbert Dijkhuizen, Sander S. van LeeuwenThe
β-galactosidase enzyme from Bacillus circulans ATCC 31382 BgaD is widely used in the food industry to produce prebiotic
galactooligosaccharides (GOS). Recently, the crystal structure of
a C-terminally truncated version of the enzyme (BgaD-D) has been elucidated.
The roles of active site amino acid residues in β-galactosidase
enzyme reaction and product specificity have remained unknown. On
the basis of a structural alignment of the β-galactosidase enzymes
BgaD-D from B. circulans and BgaA from Streptococcus
pneumoniae, and the complex of BgaA with LacNAc, we identified
eight
active site amino acid residues (Arg185, Asp481, Lys487, Tyr511, Trp570,
Trp593, Glu601, and Phe616) in BgaD-D. This study reports an investigation
of the functional roles of these residues, using site-directed mutagenesis,
and a detailed biochemical characterization and product profile analysis
of the mutants obtained. The data show that these residues are involved
in binding and positioning of the substrate and thus determine the
BgaD-D activity and product linkage specificity. This study
provides detailed insights into the structure–function relationships
of the B.
circulans BgaD-D enzyme, especially regarding GOS product
linkage specificity, allowing the rational mutation of β-galactosidase
enzymes to produce specific mixtures of GOS structures.