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Glycoside hydrolase family 81 from Clostridium thermocellum (CtLam81A), Mutant E515AGlycoside hydrolase family 81 from Clostridium thermocellum (CtLam81A), Mutant E515A
Structural highlights
Publication Abstract from PubMedThe 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 ~82kDa. CtLam81A showed maximum activity against laminarin (100U/mg), followed by curdlan (65U/mg), at pH7.0 and 75 degrees C. CtLam81A displayed Km, 2.1+/-0.12mg/ml and Vmax, 109+/-1.8U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca(2+) or Mg(2+) ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 degrees C to 96 degrees C by Ca(2+) or Mg(2+) ions and decreased to 82 degrees C by EDTA, indicating that Ca(2+) and Mg(2+) 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 1.4A 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. 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.,Kumar K, Correia MAS, Pires VMR, Dhillon A, Sharma K, Rajulapati V, Fontes CMGA, Carvalho AL, Goyal A Int J Biol Macromol. 2018 Oct 1;117:890-901. doi: 10.1016/j.ijbiomac.2018.06.003., Epub 2018 Jun 2. PMID:29870811[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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