X-Ray Crystal Structure of the Multidomain Endoglucanase Cel9G from Clostridium cellulolyticum Complexed with Natural and Synthetic Cello-Oligosaccharides

Abstract

Complete cellulose degradation is the first step in the use of biomass as a source of renewable energy. To this end, the engineering of novel cellulase activity, the activity responsible for the hydrolysis of the ␤-1,4-glycosidic bonds in cellulose, is a topic of great interest. The high-resolution X-ray crystal structure of a multidomain endoglucanase from Clostridium cellulolyticum has been determined at a 1.6-Å resolution. The endoglucanase, Cel9G, is comprised of a family 9 catalytic domain attached to a family III c cellulose-binding domain. The two domains together form a flat platform onto which crystalline cellulose is suggested to bind and be fed into the active-site cleft for endolytic hydrolysis. To further dissect the structural basis of cellulose binding and hydrolysis, the structures of Cel9G in the presence of cellobiose, cellotriose, and a DP-10 thio-oligosaccharide inhibitor were resolved at resolutions of 1.7, 1.8, and 1.9 Å, respectively.Cellulases catalyze the hydrolysis of the ␤-1,4-glycosidic linkages in cellulose, the most abundant biopolymer. Varieties of microorganisms secrete these enzymes either individually or associated in a macromolecular complex referred to as the cellulosome (23). These different cellulases, acting synergistically, catalyze the complete hydrolysis of cellulose to glucose, which, under anaerobic conditions, is a highly fermentable fuel product. The ecological and economic advantages of plant biomass conversion have spawned considerable interest in the study and eventual use of recombinant cellulolytic complexes.Clostridium cellulolyticum is an anaerobic, mesophilic, soil bacterium that is able to grow on cellulose as its sole carbon source. C. cellulolyticum secretes its cellulases in the form of a cellulosomal complex that has been extensively studied biochemically and genetically (2,14). One of these cellulases, Cel9G, is a multidomain endoglucanase capable of hydrolyzing crystalline cellulose at an appreciable rate (13). The multiple domains of this cellulase include a catalytic domain (CD) responsible for the binding and hydrolysis of cellulose, a cellulose-binding domain (CBD) responsible for the attachment of the enzyme to a cellulose chain, and the dockerin domain, which is responsible for the attachment of the cellulase to the cellulosome (35). Interestingly, in the absence of cellulosomal interactions, the dockerin domain is autolyzed in a matter of a couple of weeks at 4°C. No true cellulolytic activity is attributed to the CBD and yet, in its absence, the CD of Cel9G is inactive toward carboxymethyl cellulose, Avicel, or phosphoric acid swollen cellulose (PASC), implying a possible role in catalysis (13). In addition, when expressed as a fusion protein fused to glutathione S-transferase, the CBD alone cannot bind to cellulosic substrates.Based on sequence alignment studies, the CD of Cel9G belongs to family 9 of the glycoside hydrolase enzymes and uses a single displacement hydrolytic mechanism with net inversion of the substrate's anomeric C1 carbon. Th...

Biochemical and Mutational Analyses of a Multidomain Cellulase/Mannanase from Caldicellulosiruptor bescii

“…The C. cellulolyticum and T. fusca proteins represent two types of family 9 theme B1 endoglucanases with the enzyme-cello-oligosaccharides cocrystal structures solved (36,45). The amino acid sequence alignment showed that most of the residues involved in cellulose substrate binding are well conserved in the GH9 module of CbCel9B/ Man5A (Fig.…”

“…The amino acid sequences of the GH9 catalytic module of the Clostridium cellulolyticum Cel9G (GenBank accession number AAA73868) (36) and that of the Thermobifida fusca Cel9A (GenBank accession number AAB42155) (45) (38); and AAB42155, Cel9A of Thermobifida fusca (26,45). The aligned sequences were analyzed using BOXSHADE 3.21 (http: //www.ch.embnet.org/software/BOX_form.html) with a default setting of the fraction of sequences parameter as 0.5.…”

Structure, activity, and stability of metagenome‐derived glycoside hydrolase family 9 endoglucanase with an N‐terminal Ig‐like 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. The third group includes the structures of Cel9G from C. cellulolyticum (1G87) 14 and endo/exocellulase E4 from Thermomonospora fusca (1TF4) 15 that contain a C-terminal family 3 carbohydratebinding module (CBM3) besides the catalytic domain. The structures of the catalytic domains of these GH family 9 enzymes are characterized by the (a=a) 6 -barrel fold with three acidic active site residues (two aspartate and one glutamate residues).…”

“…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).…”

Substrate Recognition and Hydrolysis by a Family 50 exo-β-Agarase, Aga50D, from the Marine Bacterium Saccharophagus degradans

“…This is not a unique observation for a glycoside hydrolase, but it is highly rare. The first example of a CBM contributing to the formation of a glycoside hydrolase active site was observed in GH9 endoglucanases, where a family 3c CBM, which is rigidly fused to the catalytic module, extends the active site along the binding face of the CBM (66,67). A more recent example is seen in the glycogen-degrading enzyme SpuA from Streptococcus pneumoniae, where the binding site of the CBM contributes to the formation of two additional active site subsites, allowing the recognition of glycogen branch points (68).…”