Binding site analysis of cellulose binding domain CBDN1 from endoglucanse C of Cellulomonas fimi by site-directed mutagenesis. Biochemistry

ABSTRACT: Endoglucanase C (CenC), a 1,4 glucanase from the soil bacterium Cellulomonas fimi, binds to amorphous cellulose via two homologous cellulose binding domains, termed CBD N1 and CBD N2 . In this work, the contributions of 10 amino acids within the binding cleft of CBD N1 were evaluated by single site-directed mutations to alanine residues. Each isolated domain containing a single mutation was analyzed for binding to an insoluble amorphous preparation of cellulose, phosphoric acid swollen Avicel (PASA), and to a soluble glucopyranoside polymer, barley -glucan. The effect of any given mutation on CBD binding was similar for both substrates, suggesting that the mechanism of binding to soluble and insoluble substrates is the same. Tyrosines 19 and 85 were essential for tight binding by CBD N1 as their replacement by alanine results in affinity decrements of approximately 100-fold on PASA, barley -glucan, and soluble cellooligosaccharides. The tertiary structures of unbound Y19A and Y85A were assessed by heteronuclear single quantum coherence (HSQC) spectroscopy. These studies indicated that the structures of both mutants were perturbed but that all perturbations are very near to the site of mutation. Cellulose is degraded to cellobiose units by the synergistic action of several enzymes, collectively termed as cellulases. Cellulases have evolved as modular enzymes; most contain a catalytic domain (CD) 1 responsible for the hydrolysis of cellulose and usually one or more cellulose binding domains (CBD) that mediate binding of the enzyme to cellulose but are devoid of hydrolytic activity. Both domain types are the focus of independent classification systems that organize CD (1-4) and CBD sequences (5-7) into separate families by sequence homology. The roles of CBDs in cellulose degradation are still being elucidated. One obvious function of these binding domains is to keep cellulases in close contact with their substrate. Removing a CBD severely impairs the activity of a cellulase on insoluble substrates but has little effect on the degradation of soluble substrates (8-13). This suggests that the presence of a CBD is important for the efficient degradation of natural cellulose. CBDs may also be involved in the disruption of cellulose fibers by nonhydrolytic means The structures of seven CBDs are known. These include the crystal structures of CBD CipB from Clostridium thermocellum (16) and CBD E4 from Thermomonospora fusca (17), and the NMR structures of CBD CBHI from Trichoderma reseei (18), CBD Cex (19), CBD N1 (20), and CBD N2 (59) from Cellulomonas fimi, and CBD EGZ from Erwinia chrysanthemi (21). The CBDs from CBHI, Cex, E4, CipB, and EGZ, representing CBD families I, II, III, and V, respectively, all show a planar surface that is responsible for binding to crystalline cellulose. Surface-exposed aromatic residues, flanked by additional polar amino acid side chains, are present on each of the proposed binding surfaces. Sitedirected mutagenesis has been used to confirm that tyrosines are critical for the binding of family I CBD CBHI to crystalline cellulose Endoglucanase C from Cellulomonas fimi contains two family IV CBDs, CBD N1 and CBD N2 , that are present in tandem at its N-terminus. The structures of CBD N1 and CBD N2 differ from most other CBDs as they contain a binding cleft rather than a planar binding face (28). CBD N