Structure of the catalytic domain of theClostridium thermocellumcellulase CelT

Abstract

Cellulases hydrolyze cellulose, a major component of plant cell walls, to oligosaccharides and monosaccharides. Several Clostridium species secrete multi-enzyme complexes (cellulosomes) containing cellulases. C. thermocellum CelT, a family 9 cellulase, lacks the accessory module(s) necessary for activity, unlike most other family 9 cellulases. Therefore, characterization of the CelT structure is essential in order to understand its catalytic mechanism. Here, the crystal structure of free CelTΔdoc, the catalytic domain of CelT, is reported at 2.1 Å resolution. Its structure differs in several aspects from those of other family 9 cellulases. CelTΔdoc contains an additional α-helix, α-helices of increased length and two additional surface-exposed β-strands. It also contains three calcium ions instead of one as found in C. cellulolyticum Cel9M. CelTΔdoc also has two flexible loops at the open end of its active-site cleft. Movement of these loops probably allows the substrate to access the active site. CelT is stable over a wide range of pH and temperature conditions, suggesting that CelT could be used to convert cellulose biomass into biofuel.

Structure, activity, and stability of metagenome‐derived glycoside hydrolase family 9 endoglucanase with an N‐terminal Ig‐like domain

“…These structures can be divided into three groups based on the difference in the number and types of the domains. The first group includes the structures of Cel9M from Clostridium cellulolyticum (PDB ID 1IA6), 7 CelT from C. thermocellum (2YIK), 8 EF-EG2 from earthworm Eisenia fetida (3WC3), 9 and NtEgl from termite Nasutitermes takasagoensis (1KSC), 10 that only contain the catalytic domain. The second group includes the structures of Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) (3EZ8), 11 CelD from C. thermocellum (CtCelD) (1CLC), 12 and cellobiohydrolase CbhA from C. thermocellum (CtCbhA) (1UT9), 13 that contain an N-terminal immunoglobulin-like (Ig-like) domain besides the catalytic domain.…”

“…18 LC-CelG is composed of 577 amino acid residues and contains a putative signal peptide (Residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] at the N-terminus. LC-CelG without this signal peptide consists of an N-terminal Ig-like domain (Residues 20-132) and a C-terminal catalytic domain (Residues 133-577).…”

Crystal Structures of the C‐Terminally Truncated Endoglucanase Cel9Q from Clostridium thermocellum Complexed with Cellodextrins and Tris

“…The aromatic residue Trp209 (PDB ID: 1JS4) is located in subsite −3 of the processive cellulase Tf Cel9A, similar to Trp209 (PDB ID: 2XFG) in Ct Cel9I, Trp230 (PDB ID: 4DOD) in Caldicellulosiruptor bescii Cb CelA, and His220 in Ct Cel9Q (this study), whereas the corresponding residue Thr207 (PDB ID: 1IA6) in the nonprocessive cellulase Cc Cel9M is located toward subsite −4, similar to Thr356 (PDB ID: 1CLC) in Ct Cel9D, Asp302 (PDB ID: 3EZ8) in Aa Cel9A, and Ser276 (PDB ID: 2YIK) in Ct CelT (Figure ). This key residue has been proposed to distinguish processive cellulases—if the residue forms a stacking interaction with the substrate—from nonprocessive cellulases . As an exception, Cc Cel9G is a processive cellulase, but the corresponding residue is Ser208 (PDB ID: 1G87), which is not aromatic .…”

Polarity Alteration of a Calcium Site Induces a Hydrophobic Interaction Network and Enhances Cel9A Endoglucanase Thermostability

“…In contrast, the cold-adapted endoglucanase from Eisenia fetida (3WC3) has optimal activity at 40°C (43,44). In addition, the optimal temperature of CelT from Clostridium thermocellum (2YIK) is 70°C (45), which is the same as that of AaCel9A. The regions corresponding to the AaCel9A Ca-B site in 3WC3, 4DOD, and 2YIK show a significant difference in flexibility based on their B factors (Fig.…”