Roles of the catalytic domain and two cellulose binding domains of Thermomonospora fusca E4 in cellulose hydrolysis

Abstract

Thermomonospora fusca E4 is an unusual 90.4-kDa endocellulase comprised of a catalytic domain (CD), an internal family IIIc cellulose binding domain (CBD), a fibronectinlike domain, and a family II CBD. Constructs containing the CD alone (E4-51), the CD plus the family IIIc CBD (E4-68), and the CD plus the fibronectinlike domain plus the family II CBD (E4-74) were made by using recombinant DNA techniques. The activities of each purified protein on bacterial microcrystalline cellulose (BMCC), filter paper, swollen cellulose, and carboxymethyl cellulose were measured. Only the whole enzyme, E4-90, could reach the target digestion of 4.5% on filter paper. Removal of the internal family IIIc CBD (E4-51 and E4-74) decreased activity markedly on every substrate. E4-74 did bind to BMCC but had almost no hydrolytic activity, while E4-68 retained 32% of the activity on BMCC even though it did not bind. A low-activity mutant of one of the catalytic bases, E4-68 (Asp55Cys), did bind to BMCC, although E4-51 (Asp55Cys) did not. The ratios of soluble to insoluble reducing sugar produced after filter paper hydrolysis by E4-90, E4-68, E4-74, and E4-51 were 6.9, 3.5, 1.3, and 0.6, respectively, indicating that the family IIIc CBD is important for E4 processivity.

FIG. 1

Schematic diagrams of the E4 domain combinations used in this study. aa, amino acids.

FIG. 2

Viscosity reduction of CMC. Time of flow of buffer is t₀; time of flow of CMC solution is t. The concentration of E4-90, E4-68, E2, and E5 was 0.005 nmol/ml, and the concentration of E4-74 and E4-51 was 0.025 nmol/ml.

FIG. 3

Time course of products released by E4-90 digestion of SW. CB, cellobiose; Ctri, cellotriose.

FIG. 4

Binding of the E4 constructs to BMCC.

FIG. 5

Alignment of the E4 family IIIc CBD with the CipA scaffoldin CBD based on superposition of the three-dimensional structures (22, 24) by the strategy of Chothia and Lesk (6) using a 3Å cutoff to define structurally equivalent residues (denoted by black lines between the sequences). Residues where the structures diverge in their Cα placement are bracketed to emphasize that the alignment in these regions is not meaningful. Gaps are indicated by hyphens. The β strands are labeled above or below each sequence. The E4 residues which are double underlined, Asn470, Glu478, Lys480, Arg557, Glu559, Gln561, and Arg563, are conserved in family IIIc and lie in a region (colored red in Fig. 5a of reference 22) that could interact well with an extended cellulose chain from the active site. CipA residues which were mutated to Ala by Yaron et al. (25) are double underlined, and those which resulted in 3- to 10-fold lower binding to cellulose are marked by arrows. Residues marked by asterisks have the same Cα orientation, but the directions of their side chains are markedly different; the CipA Arg112 side chain is involved in a salt bridge with an Asp, while the corresponding E4 Arg563 is not. Bayer et al. (2) have identified six well-conserved residues of family III CBDs that form a shallow groove on the opposite side of the molecule from the “binding” residues; these are represented by vertical lines between the two sequences.