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Novel insights into the degradation of β-1,3-glucans by the cellulosome of Clostridium thermocellum revealed by structure and function studies of a family 81 glycoside hydrolase
- Kumar, Krishan, Correia, Márcia A.S., Pires, Virgínia M.R., Dhillon, Arun, Sharma, Kedar, Rajulapati, Vikky, Fontes, Carlos M.G.A., Carvalho, Ana Luísa, Goyal, Arun
- International journal of biological macromolecules 2018 v.117 pp. 890-901
- Clostridium thermocellum, EDTA (chelating agent), beta-glucanase, beta-glucans, calcium, catalytic activity, cellulosome, crystals, curdlan, genes, glycosides, ions, magnesium, matrix-assisted laser desorption-ionization mass spectrometry, melting curve analysis, melting point, molecular weight, mutants, pH, polymerization, site-directed mutagenesis
- The family 81 glycoside hydrolase (GH81) from Clostridium thermocellum is a β-1,3-glucanase belonging to cellulosomal complex. The gene encoding GH81 from Clostridium thermocellum (CtLam81A) was cloned and expressed displaying a molecular mass of ~82 kDa. CtLam81A showed maximum activity against laminarin (100 U/mg), followed by curdlan (65 U/mg), at pH 7.0 and 75 °C. CtLam81A displayed Km, 2.1 ± 0.12 mg/ml and Vmax, 109 ± 1.8 U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca²⁺ or Mg²⁺ ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 °C to 96 °C by Ca²⁺ or Mg²⁺ ions and decreased to 82 °C by EDTA, indicating that Ca²⁺ and Mg²⁺ ions may be involved in catalysis and in maintaining structural integrity. TLC and MALDI-TOF analysis of β-1,3-glucan hydrolysed products released initially, showed β-1,3-glucan-oligosaccharides degree of polymerization (DP) from DP2 to DP7, confirming an endo-mode of action. The catalytically inactive mutant CtLam81A-E515A generated by site-directed mutagenesis was co-crystallized and tetragonal crystals diffracting up to 1.4 Å resolution were obtained. CtLam81A-E515A contained 15 α-helices and 38 β-strands forming a four-domain structure viz. a β-sandwich domain I at N-terminal, an α/β-domain II, an (α/α)6 barrel domain III, and a small 5-stranded β-sandwich domain IV.