Probing the role of tryptophan residues in a cellulose‐binding domain by chemical modification

Abstract

The cellulose-binding domain (CBD,,,) of the mixed function glucanase-xylanase Cex from Cellulomonas fimi contains five tryptophans, two of which are located within the p-barrel structure and three exposed on the surface (Xu GY et al., 1995, Biochemistry 346993-7009). Although all five tryptophans can be oxidized by N-bromosuccinimide (NBS), stopped-flow measurements show that three tryptophans react faster than the other two. NMR analysis during the titration of CBDcex with NBS shows that the tryptophans on the surface of the protein are fully oxidized before there is significant reaction with the two buried tryptophans. Additionally, modification of the exposed tryptophans does not affect the conformation of the backbone of CBDcex, whereas complete oxidation of all five tryptophans denatures the polypeptide. The modification of the equivalent of one and two tryptophans by NBS reduces binding of CBDcex to cellulose by 70% and 90%, respectively. This confirms the direct role of the exposed aromatic residues in the binding of CBDcex to cellulose. Although adsorption to cellulose does afford some protection against NBS, as evidenced by the increased quantity of NBS required to oxidize all of the tryptophan residues, the polypeptide can still be oxidized completely when adsorbed. This suggests that, whereas the binding appears to be irreversible overall [Ong E et al., 1989, Bio/Technology 76044071, each of the exposed tryptophans interacts reversibly with cellulose.

The binding specificity and affinity determinants of family 1 and family 3 cellulose binding modules

“…This finding is in contrast to earlier data showing that isolated Cel6A CBM1 from T. reesei and some bacterial CBMs show irreversible binding at 4°C (10,18,26,27). However, when bound to crystalline cellulose, the CBM2a from Cellulomonas fimi is apparently able to diffuse laterally on the cellulose surface (28) and the tryptophans on its binding face are accessible to oxidation with N-bromosuccinimide (26). Even in the case of the Cel6A CBM1, the irreversible binding behavior seems to be borderline behavior, which can be reversed by small changes in the CBM1 structure, the binding conditions, or substrate (10).…”

“…Consistent with earlier investigations (7,12,25,26), our current data confirm that tryptophan residues contribute higher energy of binding than tyrosines on a CBM binding surface. However, the effect is limited to one Trp residue; addition of two Trp residues led to no further improvement in binding.…”

Cellulose Binding Domains of a Phytophthora Cell Wall Protein Are Novel Pathogen-Associated Molecular Patterns

“…The family 1 carbohydrate binding modules (Pfam; PF00734) represented in CBEL by two CBDs are known to occur as structurally independent, well-defined, and very compact stable domains in most fungal glycanases (Linder et al, 1995a(Linder et al, , 1995bBray et al, 1996). Importantly, CBM_1 is found in most fungi and has not been detected in higher plants (http://afmb.cnrs-mrs.fr/ CAZY/CBM_1.html).…”

An unusual choanoflagellate protein released by Hedgehog autocatalytic processing

“…These exposed Trp residues correspond to Trp259 and Trp291 in the XBD of Cellulomonas fimi xylanase D (numbering follows Simpson et al 1999) and to Trp17 and Trp54 of C. fimi Cex (numbering follows Bray et al 1996). These residues are involved in binding to xylan and to cellulose, respectively.…”