Yin Huifang, Pijning Tjaard, Meng Xiangfeng, Dijkhuizen Lubbert, van Leeuwen Sander S
Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen , Nijenborgh 7, 9747 AG Groningen, The Netherlands.
Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen , Nijenborgh 7, 9747 AG Groningen, The Netherlands.
Biochemistry. 2017 Jun 20;56(24):3109-3118. doi: 10.1021/acs.biochem.7b00207. Epub 2017 Jun 5.
The 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.
来自环状芽孢杆菌ATCC 31382的β-半乳糖苷酶BgaD在食品工业中被广泛用于生产益生元低聚半乳糖（GOS）。最近，该酶的C端截短版本（BgaD-D）的晶体结构已被阐明。活性位点氨基酸残基在β-半乳糖苷酶反应和产物特异性中的作用仍不清楚。基于环状芽孢杆菌的β-半乳糖苷酶BgaD-D与肺炎链球菌的BgaA的结构比对，以及BgaA与乳糖-N-乙酰基神经氨酸的复合物，我们在BgaD-D中鉴定出八个活性位点氨基酸残基（Arg185、Asp481、Lys487、Tyr511、Trp570、Trp593、Glu601和Phe616）。本研究报告了利用定点诱变对这些残基的功能作用进行的研究，以及对所得突变体的详细生化特性分析和产物谱分析。数据表明，这些残基参与底物的结合和定位，从而决定BgaD-D的活性和产物连接特异性。本研究提供了对环状芽孢杆菌BgaD-D酶结构-功能关系的详细见解，特别是关于GOS产物连接特异性，这使得能够对β-半乳糖苷酶进行合理突变以产生特定结构的GOS混合物。
