title: Structural insights into the substrate specificity of a 6-phospho-β-glucosidase BglA-2 from Streptococcus pneumoniae TIGR4.
abstract: The 6-phospho-β-glucosidase BglA-2 (EC 3.2.1.86) from glycoside hydrolase family 1 (GH-1) catalyzes the hydrolysis of β-1,4-linked cellobiose 6-phosphate (cellobiose-6'P) to yield glucose and glucose 6-phosphate. Both reaction products are further metabolized by the energy-generating glycolytic pathway. Here, we present the first crystal structures of the apo and complex forms of BglA-2 with thiocellobiose-6'P (a non-metabolizable analog of cellobiose-6'P) at 2.0 and 2.4 Å resolution, respectively. Similar to other GH-1 enzymes, the overall structure of BglA-2 from Streptococcus pneumoniae adopts a typical (β/α)8 TIM-barrel, with the active site located at the center of the convex surface of the β-barrel. Structural analyses, in combination with enzymatic data obtained from site-directed mutant proteins, suggest that three aromatic residues, Tyr(126), Tyr(303), and Trp(338), at subsite +1 of BglA-2 determine substrate specificity with respect to 1,4-linked 6-phospho-β-glucosides. Moreover, three additional residues, Ser(424), Lys(430), and Tyr(432) of BglA-2, were found to play important roles in the hydrolytic selectivity toward phosphorylated rather than non-phosphorylated compounds. Comparative structural analysis suggests that a tryptophan versus a methionine/alanine residue at subsite -1 may contribute to the catalytic and substrate selectivity with respect to structurally similar 6-phospho-β-galactosidases and 6-phospho-β-glucosidases assigned to the GH-1 family.
authors.affiliation: Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei Anhui 230027, China.
authors.forename: Wei-Li
authors.initials: WL
authors.lastname: Yu
authors.forename: Yong-Liang
authors.initials: YL
authors.lastname: Jiang
authors.forename: Andreas
authors.initials: A
authors.lastname: Pikis
authors.forename: Wang
authors.initials: W
authors.lastname: Cheng
authors.forename: Xiao-Hui
authors.initials: XH
authors.lastname: Bai
authors.forename: Yan-Min
authors.initials: YM
authors.lastname: Ren
authors.forename: John
authors.initials: J
authors.lastname: Thompson
authors.forename: Cong-Zhao
authors.initials: CZ
authors.lastname: Zhou
authors.forename: Yuxing
authors.initials: Y
authors.lastname: Chen
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005959:Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D005959:Glucosidases; C066586:6-phospho-beta-glucosidase
keywords: 6-Phospho-β-glucosidase; Crystal Structure; Enzyme Catalysis; Enzyme Mechanisms; PEP-PTS; Pneumonia; Streptococcus; Substrate Specificity; Thiocellobiose 6-Phosphate