jecorina and similar organisms, a model of cellulose deconstruction by cellobiohydrolases (CBHs), 6 endoglucanases, and β-glucosidases emerged ( 8). The primary organism studied for enzymatic breakdown of biomass to date is the ascomycete fungus, Hypocrea jecorina ( Trichoderma reesei) ( 6, 7).

Enzyme cocktails from fungi and bacteria, the primary degraders of plant biomass in nature, have been widely studied for biomass degradation potential, often with the aim to harness these enzymes in support of renewable fuels portfolios ( 2, 4, 5). Plants comprise the majority of terrestrial, organic carbon on earth, and organisms across all kingdoms of life have developed myriad strategies to depolymerize plant cell wall polymers for food ( 1– 3). Overall, this study reveals new structural details of GH6 CBHs likely important for functional differences among enzymes from this important family. 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. 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.

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”. One structure exhibits crystallographic waters that enable construction of a model of the α-anomer product after hydrolysis. 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. 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 bacterial Thermobifida fusca Cel6B ( TfuCel6B) exhibits a longer and more enclosed active site tunnel than its fungal counterparts. Glycoside hydrolase Family 6 (GH6) CBHs act from nonreducing ends by an inverting mechanism and are present in many cellulolytic fungi and bacteria. Their active sites are enclosed in a tunnel, enabling processive hydrolysis of cellulose chains.

Cellobiohydrolases (CBHs) are typically major components of natural enzyme cocktails for biomass degradation.

What does tfu mean