A binding‐site‐deficient, catalytically active, core protein of endoglucanase III from the culture filtrate of Trichoderma reesei

Abstract

From the culture filtrate of Trichoderma reesei we have isolated a novel endoglucanase (38 kDa) which was shown to be identical to endoglucanase I11 (E 111, 50 kDa), but lacking the first 61 N-terminal amino acids. This core protein, designated E 111 core, is fully active against soluble substrates, such as carboxymethylcellulose, whereas both activity against and adsorption to microcrystalline cellulose (Avicel) is markedly decreased. Sedimentation velocity experiments revealed that the intact E 111 enzyme has much higher assymmetry than the E 111 core protein, suggesting that the N-terminal region split off constitutes a protruding part of the native enzyme. These results lead to the proposal that native E 111 consists of two functionally separated domains: a catalytically active core and a protruding N-terminal domain which acts in the binding to insoluble cellulose. The N-terminal peptide missing in E I11 core corresponds to the heavily glycosylated common structural element found also in the N-terminus of cellobiohydrolase I1 and in the C-termini of cellobiohydrolase I and endoglucanase I. A similar bifunctional organization could thus be the rule for Trichoderma cellulases, endoglucanases as well as cellobiohydrolases.The filamentous fungus Trichoderma reesei is one of the most potent cellulose degrading organisms known [l, 21. It excretes large amounts of different cellulases which, through synergistic action [3, 41, cooperate to solubilize crystalline cellulose efficiently. Three different types of enzymes are involved in the process: (a) endo-1 ,CP-~-glucanases (EC 3.2.1.4), (b) exo-1,4-P-~-glucanases (EC 3.2.1.91) (also called cellobiohydralases) and (c) 1,4-P-~-ghcosidases (EC 3.2.1.21) [l]. Endoglucanases cut internal ~-1,4-~-glucosidic bonds in the cellulose chains. They are also able to degrade substituted cellulose such as carboxymethylcellulose, which is often used as substrate to measure endoglucanase activity. Cellobiohydrolases split off cellobiose units from the non-reducing ends of the cellulose chains. Finally, the cellobiose is hydrolyzed to glucose by the action of P-glucosidase.The exact number of enzymes excreted and their individual roles in the solubilization of cellulose is still far from clear. The picture is complicated due to the use of different growth conditions, which results in variable expression of secreted proteins, and to the existence of structural heterogeneity among several cellulases. As we shall show later on in this article, partial proteolysis of cellulases can produce fragments with full or partially impaired catalytic activity. The purification of several cellulases has been reported [5 -71. ApartCorrespondence to G. Pettersson, Institutionen for Naturvetenskaplig Biokemi, Biomedicinskt Centrum, Box 576, S-75123 Uppsala, SwedenAbbreviations. CM-cellulose, carboxymethylcellulose; CPB and CPY, carboxypeptidase B and Y; E I and E 111, endoglucanase I and 111; CBH I and CBH 11, cellobiohydrolase I and 11.Enzymes. Carboxypeptidase B (EC 3.4.17.2); carboxypeptidase Y (E...

Isolated fungal cellulose terminal domains and a synthetic minimum analogue bind to cellulose

“…The role of an extra binding region, flexibly connected to the catalytic enzyme 'core' is not clear, but comparison between T. reesei EIII with and without the A-B-region shows that the difference in catalytic efficiency toward Avicel parallels the difference in binding to the substrate [9]. A similar enhancement of the binding efficiency involving only the catalytic site would have a negative effect on the turnover number of the hydrolysis.…”

“…Physicochemical studies on enzymes with and without a B-Aregion [7,8] show that it constitutes a protruding part of the intact enzyme and can be regarded as Abbreviations: Tr, Trichoderma reesei; Sp, Sporotrichum pulverulentum; Boc, t-butyioxycarbonyi; Bzl, benzyl; Br-Z, 2-bromobenzyloxycarbonyl; Me-Bzl, 4-methylbenzyl; Dnp, 2,4-dinitrophenyl; DMF, dimethyl formamide; DCM, dichloromethane; TFA, trifluoroacetic acid a separate domain. The role of the whole B-Aregion is to enhance the activity towards the solid substrate [9][10][11]. The importance of this structural organization is demonstrated by the fact that it also occurs in two cellulases from the unrelated bacterium Cellulomonas fimi [12].…”

Origin of Initial Burst in Activity for Trichoderma reesei endo-Glucanases Hydrolyzing Insoluble Cellulose

“…TrCel7B, TrCel5A, and TrCel12A (4) belong to glycoside hydrolase families 7 (15), 5 (16), and 12 (17), respectively. TrCel7B has a C-terminal carbohydrate binding module I and shares 40 -45% overall homology to the exo-cellulase TrCel7A (18,19). TrCel5A has the least homology to the other T. reesei cellulases and has an N-terminal carbohydrate binding module I (16).…”

Three‐dimensional structures of three engineered cellulose‐binding domains of cellobiohydrolase I from Trichoderma reesei

“…Thus, CBD is required for the full activity of cellulases on crystalline cellulose (van Tilbeurgh et al, 1986;Stihlberg et al, 1988Stihlberg et al, , 1991., 1988; Reinikainen et al, 1992, Structureactivity relationship of engineered CBDcBHl 1995), however, the detailed binding mechanism of the CBD is not known.…”