The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose.

Abstract

Cellulose-binding domains (CBDs) bind specifically to cellulose, and form distinct domains of most cellulose degrading enzymes. The CBD-mediated binding of the enzyme has a fundamental role in the hydrolysis of the solid cellulose substrate. In this work we have investigated the reversibility and kinetics of the binding of the CBD from Trichoderma reesei cellobiohydrolase I on microcrystalline cellulose. The CBD was produced in Escherichia coli, purified, and radioactively labeled by reductive alkylation with 3H.Sensitive detection of the labeled CBD allowed more detailed analysis of its behavior than has been possible before, and important novel features were resolved. Binding of the CBD was found to be temperature sensitive, with an increased affinity at lower temperatures. The interaction of the CBD with cellulose was shown to be fully reversible and the CBD could be eluted from cellulose by simple dilution. The rate of exchange measured for the CBD-cellulose interaction compares well with the hydrolysis rate of cellobiohydrolase I, which is consistent with its proposed mode of action as a processive exoglucanase.Cellulose is a polymer composed of D-glucose units linked by 13-1,4-glycosidic bonds. The cellulose chains can pack together to form fibers, which are crystalline in nature (1). This solid and ordered substrate poses some special requirements on the enzymes hydrolyzing it. Most cellulose degrading enzymes have a bifunctional organization composed of Iwo domains connected by a linker region. One of the domaind is dedicated for binding to the solid cellulose and is called a cellulosebinding domain (CBD). The other domain contains the catalytic machinery. These catalytic domains have been grouped into several families sharing the same basic structure and the same chemical reaction mechanism (2). Trichoderma reesei cellobiohydrolase I (CBHI) is an exoglucanase with a long tunnel-shaped active site (3). CBHI and other exoglucanases initiate the hydrolysis at the chain ends, and do not produce significant amounts of new chain ends on the cellulose surface (4). They seem to act by a processive mechanism hydrolyzing consecutive bonds along the same chain without dissociating (2, 3). The endoglucanases cleave internal bonds along the cellulose chains probably by cycles of adsorption and desorption.Based on sequence comparisons the CBDs have been grouped into several families (5). To date, the threedimensional structures of two CBDs have been published. One is from T. reesei CBHI (6) and the other from Cellulomonas fimi xylanase/cellobiohydrolase Cex (7). Both the fold and the size of these CBDs are different, but a common pattern of aromatic residues and amides is found at the surface of both proteins. Many experiments have shown that this surface is the primary interaction site between a CBD and cellulose (7-10). Many studies directly correlate the efficiency of crystalline cellulose degradation to the binding efficiency of the enzyme (11) and the removal of a CBD typically results in a decrease of a...

Processivity of Cellobiohydrolases Is Limited by the Substrate

“…For both TrCel7A and PcCel7D, the k off values on rBC were about 3 times lower than on rAC, indicating that interactions between the cellulose chain and the active site tunnel of CBHs are not sole determinants of k off . The k off value of 0.029 Ϯ 0.003 s Ϫ1 (30°C) has been reported for dissociation of isolated carbohydrate binding module of TrCel7A from bacterial microcrystalline cellulose (50). This figure is about 30 times higher than the k off found for dissociation of TrCel7A from rBC, suggesting that dissociation of TrCel7A is not limited by the dissociation of its carbohydrate binding module.…”

Specificity of O -glycosylation in enhancing the stability and cellulose binding affinity of Family 1 carbohydrate-binding modules

“…Subsequently, we synthesized two additional series of CBMs that had specific glycans at more than one of three glycosylation sites (11)(12)(13)(14)(15)(16)(17)(18)(19)(20) (Fig. 2).…”

“…Alternatively, O-linked glycans distant from the binding face of a Family 2a CBM do not affect cellulose affinity (43), and large high mannose-type N-linked glycans near the Family 2a CBM binding face detrimentally affect cellulose affinity (44). Family 1 CBM experimental studies to date have examined the functional role of many structural features, but no work has systematically considered the effects of the natural O-mannosylation (9,12,13,15,43,45). To address the various potential effects of O-mannosylation on the TrCel7A CBM, we performed the comparative study using synthetic homogenous glycoforms.…”

“…The structure of the TrCel7A CBM revealed a β-sheet-rich structure with two disulfide bridges and a flat face decorated with aromatic and polar residues that form the putative binding face for adsorption to the hydrophobic face of crystalline cellulose microfibrils ( Fig. 1) (11)(12)(13)(14)(15).…”

Systems analysis of plant cell wall degradation by the model filamentous fungus Neurospora crassa

“…Many plant cell wall degrading enzymes contain a cellulosebinding module (CBM), which aids in attachment of the enzyme to the substrate (17). Within the N. crassa genome, 19 genes are predicted to encode proteins with a CBM1 domain (18).…”