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

DOI: 10.1016/j.ijbiomac.2018.06.003

Authors: Krishan Kumar (Indian Institute of Technology Guwahati) , Marcia Correia (UCIBIO-REQUIMTE, Universidade Nova de Lisboa) , Virgínia R. Pires (Universidade de Lisboa) , Arun Dhillon (Indian Institute of Technology Guwahati) , Kedar Sharma (Indian Institute of Technology Guwahati) , Vikky Rajulapati (Indian Institute of Technology Guwahati) , Carlos M. G. A. Fontes (Universidade de Lisboa; NZYTech - Genes & Enzymes) , Ana Luisa Carvalho (UCIBIO-REQUIMTE, Universidade Nova de Lisboa) , Arun Goyal (Indian Institute of Technology Guwahati)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: International Journal Of Biological Macromolecules

State: Published (Approved)
Published: June 2018
Diamond Proposal Number(s): 16609

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 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.

Journal Keywords: Clostridium thermocellum; β-1,3-Glucanase; Laminarin; Thin layer chromatography; X-ray crystallography

Diamond Keywords: Bacteria; Enzymes

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I04-Macromolecular Crystallography

Added On: 06/06/2018 08:30

Discipline Tags:

Biochemistry Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)