title: Characterization of a novel metagenome-derived 6-phospho-β-glucosidase from black liquor sediment.
abstract: The enzyme 6-phospho-β-glucosidase is an important member of the glycoside hydrolase family 1 (GH1). However, its catalytic mechanisms, especially the key residues determining substrate specificity and affinity, are poorly understood. A metagenome-derived gene sequence, encoding a novel 6-phospho-β-glucosidase designated Pbgl25-217, was isolated and characterized. The optimal conditions for enzymatic activity were 37°C and pH 7; Ca(2+), Mg(2+), and Mn(2+) stabilized the activity of Pbgl25-217, whereas Ni(2+), Fe(2+), Zn(2+), Cu(2+), and Fe(3+) inhibited its activity. The Km and Vmax of Pbgl25-217 were 4.8 mM and 1,987.0 U mg(-1), respectively. Seven conserved residues were recognized by multiple alignments and were tested by site-directed mutagenesis for their functions in substrate recognition and catalytic reaction. The results suggest that residues S427, Lys435, and Tyr437 act as "gatekeepers" in a phosphate-binding loop and play important roles in phosphate recognition. This functional identification may provide insights into the specificity of 6-phospho-β-glycosidases in GH1 and be useful for designing further directed evolution.
authors: State Key Laboratory of Microbial Technology, Shandong University, Jinan, People's Republic of China.
authors: Chunyu
authors: C
authors: Yang
authors: Yu
authors: Y
authors: Niu
authors: Chunfang
authors: C
authors: Li
authors: Deyu
authors: D
authors: Zhu
authors: Wei
authors: W
authors: Wang
authors: Xinqiang
authors: X
authors: Liu
authors: Bin
authors: B
authors: Cheng
authors: Cuiqing
authors: C
authors: Ma
authors: Ping
authors: P
authors: Xu
mesh_terms: D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D020536:Enzyme Activators / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D005959:Glucosidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology; D007700:Kinetics; D054892:Metagenome; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D005959:Glucosidases; C066586:6-phospho-beta-glucosidase