Loop Motions Important to Product Expulsion in the Thermobifida fusca Glycoside Hydrolase Family 6 Cellobiohydrolase from Structural and Computational Studies

Miao Wu,Lintao Bu,Thu V Vuong,David B Wilson,Michael F Crowley,Mats Sandgren,Jerry Ståhlberg,Gregg T Beckham,Henrik Hansson

doi:10.1074/jbc.m113.502765

Abstract

Cellobiohydrolases (CBHs) are typically major components of natural enzyme cocktails for biomass degradation. Their active sites are enclosed in a tunnel, enabling processive hydrolysis of cellulose chains. Glycoside hydrolase Family 6 (GH6) CBHs act from nonreducing ends by an inverting mechanism and are present in many cellulolytic fungi and bacteria. The bacterial Thermobifida fusca Cel6B (TfuCel6B) exhibits a longer and more enclosed active site tunnel than its fungal counterparts. Here, we determine the structures of two TfuCel6B mutants co-crystallized with cellobiose, D274A (catalytic acid), and the double mutant D226A/S232A, which targets the putative catalytic base and a conserved serine that binds the nucleophilic water. The ligand binding and the structure of the active site are retained when compared with the wild type structure, supporting the hypothesis that these residues are directly involved in catalysis. One structure exhibits crystallographic waters that enable construction of a model of the α-anomer product after hydrolysis. Interestingly, the product sites of TfuCel6B are completely enclosed by an "exit loop" not present in fungal GH6 CBHs and by an extended "bottom loop". From the structures, we hypothesize that either of the loops enclosing the product subsites in the TfuCel6B active site tunnel must open substantially for product release. With simulation, we demonstrate that both loops can readily open to allow product release with equal probability in solution or when the enzyme is engaged on cellulose. Overall, this study reveals new structural details of GH6 CBHs likely important for functional differences among enzymes from this important family.

Highlights

6 glycoside hydrolases represent an important, diverse enzyme class in cellulolytic organisms
We examined whether substrate complexation on the cellulose surface can block either of the loops opening by running an molecular dynamics (MD) simulation (Fig. 3) to model the post-hydrolysis system with a cellobiose in the Ϫ1 and Ϫ2 subsites and a cellotetraose spanning the ϩ4 to ϩ1 subsites, which is further bonded to the cellulose chain being decrystallized from the crystal surface
Our results suggest that catalytic engagement of TfuCel6B on a cellulose surface may influence the flexibility of the tunnel-forming loops in TfuCel6B differently from that in solution, and product expulsion was examined in both cases

Summary

Background

6 glycoside hydrolases represent an important, diverse enzyme class in cellulolytic organisms. Cellobiohydrolases (CBHs) are typically major components of natural enzyme cocktails for biomass degradation Their active sites are enclosed in a tunnel, enabling processive hydrolysis of cellulose chains. TfuCel6B is an interesting GH6 model system because of its extended and more enclosed tunnel, and because Wilson and co-workers [33, 34] have conducted considerable experimental work on its catalytic mechanism, substrate specificity, and synergy with other T. fusca enzymes, providing significant mutational data for mechanistic interpretations. Based on the new structures presented here and the previously published wild type structures [23], we conduct various molecular dynamics (MD) simulations to examine questions of enzyme and ligand dynamics relative to HjeCel6A, cellobiose product release after hydrolysis, and the complexation of the TfuCel6B catalytic domain on the surface of a cellulose I␤ crystal

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION