Abstract：

The surface-exposed β-galactosidase BgaC from Streptococcus pneumoniae was reported to be a virulence factor because of its specific hydrolysis activity towards the β(1,3)-linked galactose and N-acetylglucosamine [Galβ(1,3)NAG] moiety of oligosaccharides on the host molecules. Here we report the crystal structure of BgaC at 1.8 Ǻ and its complex with galactose at 1.95 Ǻ. At pH 5.5 to 8.0, BgaC exists as a stable homodimer, each subunit of which consists of three distinct domains: a catalytic domain of a classic (β/α)8 TIM barrel, followed by two all-β domains (ABDs) of unknown function. The side-walls of the TIM β-barrel and a loop extended from the first ABD constitute the active site. Superposition of the galactose-complexed structure to the apo-form revealed significant conformational changes of residues Trp243 and Tyr455. Simulation of a putative substrate entrance tunnel and modeling of a complex structure with Galβ(1,3)NAG enabled us to assign three key residues to the specific catalysis. Site-directed mutagenesis in combination with activity assays further proved that residues Trp240 and Tyr455 contribute to stabilizing the N-acetylglucosamine moiety, whereas Trp243 is critical for fixing the galactose ring. Moreover, we propose that BgaC and other galactosidases in the GH-35 family share a common domain organization and a conserved substrate-determinant aromatic residue protruding from the second domain.