Abstract
Trichoderma reesei Cel6A (TrCel6A) is a cellobiohydrolase that hydrolyzes crystalline cellulose into cellobiose. Here we directly observed the reaction cycle (binding, surface movement, and dissociation) of single-molecule intact TrCel6A, isolated catalytic domain (CD), cellulose-binding module (CBM), and CBM and linker (CBM-linker) on crystalline cellulose Iα. The CBM-linker showed a binding rate constant almost half that of intact TrCel6A, whereas those of the CD and CBM were only one-tenth of intact TrCel6A. These results indicate that the glycosylated linker region largely contributes to initial binding on crystalline cellulose. After binding, all samples showed slow and fast dissociations, likely caused by the two different bound states due to the heterogeneity of cellulose surface. The CBM showed much higher specificity to the high affinity site than to the low affinity site, whereas the CD did not, suggesting that the CBM leads the CD to the hydrophobic surface of crystalline cellulose. On the cellulose surface, intact molecules showed slow processive movements (8.8 ± 5.5 nm/s) and fast diffusional movements (30–40 nm/s), whereas the CBM-Linker, CD, and a catalytically inactive full-length mutant showed only fast diffusional movements. These results suggest that both direct binding and surface diffusion contribute to searching of the hydrolysable point of cellulose chains. The duration time constant for the processive movement was 7.7 s, and processivity was estimated as 68 ± 42. Our results reveal the role of each domain in the elementary steps of the reaction cycle and provide the first direct evidence of the processive movement of TrCel6A on crystalline cellulose.
Highlights
The previous studies proposed the processive hydrolysis of Trichoderma reesei Cel7A (TrCel7A) and TrCel6A, a continuous reaction without dissociation from cellulose surface
We concluded that the slow component seen only in Intact corresponds to the processive movement accompanying the hydrolysis of crystalline cellulose, whereas the fast component is the diffusive movement on the cellulose surface without hydrolysis
We verified the processive movement of TrCel6A and clarified the kinetic role of the cellulose binding module (CBM) and glycosylated linker region using single-molecule fluorescence imaging
Summary
Hydrolysis Activities of Cy3-TrCel6A, Inactive Cy3-TrCel6A, and Cy3-CD—For single-molecule fluorescence imaging analysis, mutants of the full-length molecule (447aa), TrCel6A (S386C) and TrCel6A (D221A/S386C), isolated CD (S386C), CBM-linker (S83C), and CBM (S43C) were generated (Fig. 2A). The hydrolysis activities of Intact against crystalline cellulose I␣ were similar to those of wild-type TrCel6A (WT) at various concentrations of the substrate (Fig. 3 and Table 1). Hydrolysis activities against amorphous cellulose (phosphoric acid swollen cellulose (PASC)) were similar among the WT, Intact, and CD (0.42 Ϯ 0.03, 0.41 Ϯ 0.07, and 0.43 Ϯ 0.09 sϪ1, respectively) (Table 1). These results indicate that the CBM-linker is important for crystalline cellulose degradation. The k slow off values for Intact and the CBM were similar but less than those for the CBM-linker
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
