title: Structural insights into the substrate specificity of Streptococcus pneumoniae β(1,3)-galactosidase BgaC.
abstract: The surface-exposed β-galactosidase BgaC from Streptococcus pneumoniae was reported to be a virulence factor because of its specific hydrolysis activity toward 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 Å and its complex with galactose at 1.95 Å. At pH 5.5-8.0, 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 Trp-243 and Tyr-455. 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 Trp-240 and Tyr-455 contribute to stabilizing the N-acetylglucosamine moiety, whereas Trp-243 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.
authors.affiliation: Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China.
authors.forename: Wang
authors.initials: W
authors.lastname: Cheng
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Yong-Liang
authors.initials: YL
authors.lastname: Jiang
authors.forename: Xiao-Hui
authors.initials: XH
authors.lastname: Bai
authors.forename: Jun
authors.initials: J
authors.lastname: Chu
authors.forename: Qiong
authors.initials: Q
authors.lastname: Li
authors.forename: Ge
authors.initials: G
authors.lastname: Yu
authors.forename: Qiu-Ling
authors.initials: QL
authors.lastname: Liang
authors.forename: Cong-Zhao
authors.initials: CZ
authors.lastname: Zhou
authors.forename: Yuxing
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000502:physiology; D003461:Crystallography; D019281:Dimerization; D005690:Galactose / Q000378:metabolism*; D016296:Mutagenesis / Q000502:physiology; D017434:Protein Structure, Tertiary; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000472:pathogenicity; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014774:Virulence; D037521:Virulence Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D037521:Virulence Factors; D001616:beta-Galactosidase; D000117:Acetylglucosamine; D005690:Galactose