Novel insights into the degradation of beta-1,3-glucans by the cellulosome of Clostridium thermocellum revealed by structure and function studies of a family 81 glycoside hydrolase

摘要

The family 81 glycoside hydrolase (GH81) from Clostridium thermocellum is a beta-1,3-glucanase belonging to cellulosomal complex. The gene encoding GH81 from Clostridium thermocellum (CtLam81A) was cloned and expressed displaying a molecular mass of similar to 82 kDa. CtLam81A showed maximum activity against laminarin (100 U/mg), followed by curdlan (65 U/mg), at pH 7.0 and 75 degrees C. CtLam81A displayed K-m, 2.1 +/- 0.12 mg/ml and V-max, 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 degrees C to 96 degrees C by Ca2+ or Mg2+ ions and decreased to 82 degrees 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 beta-1,3-glucan hydrolysed products released initially, showed beta-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 angstrom resolution were obtained. CtLam81A-E515A contained 15 alpha-helices and 38 beta-strands forming a four-domain structure viz. a beta-sandwich domain I at N-terminal, an alpha/beta-domain II, an (alpha/alpha)(6) barrel domain III, and a small 5-stranded beta-sandwich domain IV. (C) 2018 Elsevier B.V. All rights reserved.