6FOP

Glycoside hydrolase family 81 from Clostridium thermocellum (CtLam81A), Mutant E515A

Summary for 6FOP

• Entry DOI: 10.2210/pdb6fop/pdb
• Descriptor: Glycoside hydrolase family 81, CALCIUM ION, (4S)-2-METHYL-2,4-PENTANEDIOL, ... (9 entities in total)
• Functional Keywords: gh81, glycoside hydrolase, clostridium thermocellum, glucanase, laminarin, hydrolase
• Biological source: Clostridium thermocellum (Ruminiclostridium thermocellum)
• Total number of polymer chains: 1
• Total formula weight: 82059.85

Authors

Correia, M.A.S.C.,Carvalho, A.L. (deposition date: 2018-02-08, release date: 2019-03-13, Last modification date: 2024-01-17)

Primary citation

Kumar, K.,Correia, M.A.S.,Pires, V.M.R.,Dhillon, A.,Sharma, K.,Rajulapati, V.,Fontes, C.M.G.A.,Carvalho, A.L.,Goyal, A.
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.
Int.J.Biol.Macromol., 117:890-901, 2018

PubMed Abstract: 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 K, 2.1 ± 0.12 mg/ml and V, 109 ± 1.8 U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca or Mg ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 °C to 96 °C by Ca or Mg ions and decreased to 82 °C by EDTA, indicating that Ca and Mg 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) barrel domain III, and a small 5-stranded β-sandwich domain IV.

PubMed: 29870811
DOI: 10.1016/j.ijbiomac.2018.06.003

Experimental method

X-RAY DIFFRACTION (1.4 Å)