Structural studies of beta-galactosidase from Bifidobacterium bifidium, a hydrolase from GH family 42

Enzymatic hydrolyses of cell wall is one of the main challenges for second-generation bioethanol production. This may be due not only by the complexity of the cell wall itself, but also for our lack of knowledge of enzymatic mechanisms. Many classes of enzymes are not completely understood, making difficult to elaborate highly efficient enzymatic cocktails. Here we studied the β-galactosidases from family 42, one of the least studied families from this class of enzymes. β-galactosidases are responsible not only for the hydrolysis of β(1-4) linkages in saccharides, but also to synthetize galactooligosaccharides, and have a wide applicability in biotechnology and food industry. To accomplish this, we have cloned the lacZ2 gene from Bifidobacterium bifidium S17, a well-adapted organism from human digestive tract. Bb_LacZ2 was expressed, purified, crystallized and characterized against synthetic and natural substrates. X-ray structure of the enzyme was solved and refined to 1.7 Å resolution. By careful investigation of Bb_LacZ2 interactions with galactose we have identified a number of residues that are involved in stabilization and the mechanism hydrolysis itself. These residues were explored by site-directed mutagenesis, which showed that Glu161 and Glu320 are, respectively, the acid/base catalyst and the nucleophile. We also revealed that Asn160 and Glu368 are fundamental for the enzyme catalytic efficiency, while Tyr289 and His371 are important for the enzyme affinity to the substrate.