Abstract
Glucosidases play key roles in many diseases and are limiting enzymes during cellulose degradation, which is an important part of global carbon cycle. Here, we identified a novel β-glucosidase, CmGH1, isolated from marine bacterium Croceicoccus marinus E4A9T. In spite of its high sequence and structural similarity with β-xylosidase family members, CmGH1 had enzymatic activity toward p-nitrophenyl-β-D-glucopyranoside (p-NPG) and cellobiose. The Km and Kcat values of CmGH1 toward p-NPG were 0.332 ± 0.038 mM and 2.15 ± 0.081 min–1, respectively. CmGH1 was tolerant to high concentration salts, detergents, as well as many kinds of organic solvents. The crystal structure of CmGH1 was resolved with a 1.8 Å resolution, which showed that CmGH1 was composed of a canonical (α/β)8-barrel catalytic domain and an auxiliary β-sandwich domain. Although no canonical catalytic triad residues were found in CmGH1, structural comparison and mutagenesis analysis suggested that residues Gln157 and Tyr264 of CmGH1 were the active sites. Mutant Q157E significantly increased its hydrolase activity up to 15-fold, whereas Y264E totally abolished its enzymatic activity. These results might provide new insights into understanding the different catalytic mechanism during evolution for β-glucosidases and β-xylosidases.
Highlights
Structural and Functional Insights Into CmGH1 and food industries (Bosetto et al, 2016)
CmGH1 was predicted as a β-xylosidase, the biochemical results suggested CmGH1 might be a β-D-glucosidase belonging to the GH39 family
The auto-docking result showed that CmGH1 preferred to combine cellobiose and glucose, which had lower binding energy and inhibition constant compared with xylobiopyranose and xylopyranose
Summary
Structural and Functional Insights Into CmGH1 and food industries (Bosetto et al, 2016). According to the amino acid sequence and structure, β-xylosidases are generally assigned to glycoside hydrolase (GH) families 1, 3, 30, 39, 43, 51, 52, 54, 116, and 120 (Henrissat and Davies, 1997). Β-glucosidases are well-characterized and generally classified into many GH families 1, 2, 3, 5, 9, 30, 39, and 116 (Henrissat and Davies, 1997). There is no β-glucosidase reported in GH39 family and the structural basis for substrate specificity is barely investigated. We present the characterizations and crystal structure of CmGH1, which showed β-glucosidase activity, instead of β-xylosidase activity, to provide a structural basis for the development, and utilization of the marine sourced β-glucosidase
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