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Structure and biochemical characterization of glucose tolerant β-1,4 glucosidase (HtBgl) of family 1 glycoside hydrolase from Hungateiclostridium thermocellum
- Sharma, Kedar, Thakur, Abhijeet, Kumar, Rajeev, Goyal, Arun
- Carbohydrate research 2019 v.483 pp. 107750
- amino acids, catalytic activity, cellobiose, crystal structure, glucose, glycosides, hydrolases, ligands, magnesium, melting, models, molecular dynamics, pH, proteins, temperature, thermal stability, triose-phosphate isomerase
- β-1,4-glucosidase (HtBgl) of family 1 glycoside hydrolase from Hungateiclostridium thermocellum was cloned in pET28a(+) vector, expressed, biochemically and structurally characterized. HtBgl displayed 67 U/mg activity against 4-nitrophenyl-β-d-glucopyranoside, followed by 180 U/mg against cellobiose and 42 U/mg activity against 4-nitrophenyl-β-d-galactopyranoside. HtBgl displayed an optimum temperature of 65 °C and an optimum pH of 6.0. HtBgl was stable in the pH range, 4.0–8.0 and displayed the thermostability up to 60 °C for 1 h. HtBgl displayed the glucose tolerance up to 750 mM and retained ~70% activity after 20 h. HtBgl crystal structure submitted (PDB id 5OGZ) by others exhibited a classical Triosephosphate Isomerase, (β/α)8-barrel fold. Protein melting analysis of HtBgl exhibited a single peak at 78 °C and the addition of 5 mM Mg2+ shifted the peak to 82 °C. Molecular dynamics studies showed that the amino acid residues from 351 to 375 exhibit the flexibility due to the presence of the catalytic acid residue. The structure comparison of HtBgl with homologous proteins and its docking analysis with probable ligands revealed that the residues, E166 and E355 are involved in the catalysis. The SAXS analysis of HtBgl showed that the protein is monomeric and present in a fully folded state. The radius of gyration (Rg) found was 2.15–2.26 nm. The bell-shaped curve obtained by Kratky plot analysis displayed the globular shape and fully folded state with flexibility in the N-terminal region. The HtBgl crystal structure superposed well with the SAXS derived dummy atom model.