title: The 184th residue of β-glucosidase Bgl1B plays an important role in glucose tolerance.
abstract: Bgl1B (ACY09072) is a new GH1 family β-glucosidase derived from a marine microbial metagenomic library. In our previous study, the activity of recombinant Bgl1B was competitively inhibited by glucose with an IC₅₀ value of 30 mM. Based on amino acids sequence alignment with other GH1 β-glucosidases, including BglB from Paenibacillus polymyxa whose structure has been solved, the 184th and 409th residues of Bgl1B were suggested to be key residues relating to glucose tolerance or other properties. The putative role of the two residues of Bgl1B was investigated by site-directed mutagenesis by replacing His184 and Leu409 with Phe and Glu, respectively. Biochemical characterization data indicated that the mutations had little influence on the enzyme properties. However, in comparison with wild-type Bgl1B, the mutant H184F exhibited lower stability at all tested temperatures and pHs, while L409E exhibited higher stability. Particularly, when using 4-Nitrophenyl-β-D-glucopyranoside (pNPG) as substrate, the mutant H184F exhibited much better glucose tolerance, with a K(i) of 76.9 mM, than L409E and wild-type Bgl1B (17.2 mM and 14.9 mM, respectively). These results indicated that the 184th residue might play an important role in the glucose tolerance of Bgl1B.
authors.affiliation: School of Life Sciences, Anhui University, Hefei, Anhui 230039, China.
authors.forename: Juanjuan
authors.initials: J
authors.lastname: Liu
authors.forename: Xuecheng
authors.initials: X
authors.lastname: Zhang
authors.forename: Zemin
authors.initials: Z
authors.lastname: Fang
authors.forename: Wei
authors.initials: W
authors.lastname: Fang
authors.forename: Hui
authors.initials: H
authors.lastname: Peng
authors.forename: Yazhong
authors.initials: Y
authors.lastname: Xiao
mesh_terms: D019943:Amino Acid Substitution; D004795:Enzyme Stability; D007700:Kinetics; D056186:Metagenomics; D016297:Mutagenesis, Site-Directed; D056507:Paenibacillus / Q000201:enzymology / Q000378:metabolism; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001617:beta-Glucosidase