Article Figures & Data
Figures
- FIG. 1.
Space-filled structure of Cel9A-68 (4TF4) with six glucose molecules in the catalytic cleft. This enzyme product structure was obtained after soaking the crystals in cellopentaose (G5). G5 was cleaved to G4 and G1 or G3 and G2, and density for six glucose residues can be seen in the structure because G5 can bind from the position of Glc(−3) to Glc(+2) or from Glc(−4) to Glc(+1). The CBM residues chosen for mutation are shown in dark gray.
- FIG. 2.
Cel9A-68 product complex structure (4TF4) showing amino acids chosen for mutation around the catalytic center in light gray and the six sugar residues in dark gray. Hydrogen bonds are shown as dashed lines. Cleavage occurs between Glc(−1) and Glc(+1).
- FIG. 3.
Sodium azide rescue test. The samples were treated with various concentrations of azide (0 M to 0.6 M) for 16 h, and then SWC activity was assayed and calculated [μmol of cellobiose/(min·μmol of enzyme)]. The average coefficients of variation were 4%, 5%, 3%, and 4% for wild-type and E424, D55, and D58 mutant enzymes, respectively.
- FIG. 4.
Enzyme product complex (4TF4) showing the hydrogen bond network between Y206, D55, Glc(−1), D58, and H125. The hydrogen bonds from the catalytic acid E424 to Glc(−1) and Glc(+1) are also shown.
- FIG. 5.
Structure-based sequence alignment of family 3 CBMs. Secondary structural β-strands are indicated by arrows and enumerated. Proposed cellulose binding residues are shown in dashed boxes, and for T. fusca Cel9A-68, they are F476, D513, Y520, Q561, and R563. Residues shown in solid boxes are conserved, and those T. fusca residues are V465, Y501, Y538, E559, N574, D575, S577, Y594, V600, and P605. Sequences were obtained from the respective GenBank accession codes (included parenthetically) as follows: Cel9A_Thefu, from Thermobifida fusca (M73322); CelQ_Cloth, from Clostridium thermocellum (AB047845); Cel9G_Cloce, from Clostridium cellulolyticum (M87018); EngH_Cloce, from Clostridium cellulovorans (U34793); CelZ2_Clost, from Clostridium stercorarium (X55299); Cel9I2_Cloth, from Clostridium thermocellum (L04735); Cel9B_Cloth, from Clostridium thermocellum (X60545); Cel9A_Celfi, from Cellulomonas fimi (M64644); CipC_Cloce, from Clostridium cellulolyticum (U40345); CipA1_Cloth, from Clostridium thermocellum (X67406); CbpA_Cloce, from Clostridium cellulovorans (M73817); CipA_Clojo, from Clostridium josui (AB004845); CelZ1_Clost, from Clostridium stercorarium (X55299); CelA_Calsa, from Caldicellulosiruptor saccharolyticus (M36063); CelA_Paela, from Paenibacillus lautus (M76588); and Cel9I1_Cloth, from Clostridium thermocellum (L04735).
Tables
- TABLE 1.
Activity and binding characteristics of mutant enzymes
Enzyme Activity on substratea (μmol cellobiose min−1 μmol enzyme−1): Processivityb (ratio of soluble/insoluble reducing ends produced) Binding to: CMC SWC BC FP BCb (% bound) MUG3 (dissociation constant [μM]) Cel9A-68 amino acids 1-613 Wild type 279 10.5 3.3 0.28 3.1 15 0.57 Key catalytic residue mutants E424A 0.28d 0.03d 0.01d 40 1.23d D55A 0.6d 0.14d 0.03d 2.21d D55C 9.3 0.3 32 D58A 0.9d 0.21d 0.07d 0.50d Y206S 1.0d 0.15d 0.040d 0.06d 0d 5 Poord H125N 7.9 0.4 0.11 0.07 0.3 25 H125A 6.7 0.7 0.09 0.05 0 36 0.73 CD mutants interacting with Glc(−1) to Glc(−4) F205A 96 4.4 0.33 0.14 1.4 11 2.30 Y429A 58 3.0 0.37 0.15 1.3 26 0.26 W313G 10 0.6 0.10 0.08 1.0 13 Poor W313D 8.4 0.5 0.08 0.06 0.9 16 Poor W209S 629 10.5 0.46 0.18 2.6 21 W256A 1,080 7.4 0.26 0.13 1.8 16 R317K 443 9.2 0.44 0.18 2.2 19 D261A 758 9.6 0.49 0.17 2.6 20 D261N 1,510 10.0 0.37 0.16 1.6 18 CD mutants interacting with Glc(+1), Glc(+2), and putative Glc(+3) R378K 270 9.6 3.4 0.26 4.3 20 W128A 32 0.4 0.25 0.12 1.9 18 CD double mutant D261A/R378K 503 3.0 0.18 0.10 1.3 18 CBM mutants Y520A 302 8.2 1.1 0.22 2.7 14 D513A 279 12.1 4.0 0.30 3.7 20 I514H 290 9.6 3.7 0.29 3.4 23 R557A/E559A 252 7.9 0.48 0.21 3.3 15 F476A 270 11 2.6 0.28 4.5 21 R563A 270 7.4 1.7 0.26 4.0 20 Cel9A-51 amino acids 1-463 Wild type 24 0.6 0.2c 0.13c 0.6c 8 D55C 0.9 0.1 10
