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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.