Crystal structure of the catalytic core domain of the family 6 cellobiohydrolase II, Cel6A, from Humicola insolens, at 1.92 angstrom resolution

The three-dimensional structure of the catalytic core of the family 6 cello biohydrolase II, Cel6A (CBH II), from Humicola insolens has been determined by X-ray crystallography at a resolution of 1.92 A Angstrom. The structure was solved by molecular replacement using the homologous Trichoderma reese i CBH II as a search model. The H. insolens enzyme displays a high degree o f structural similarity with its T. reesei equivalent. The structure featur es both O- (alpha-linked mannose) and N-linked glycosylation and a hexa-co- ordinate Mg2+ ion. The active-site residues are located within the enclosed tunnel that is typical for cellobiohydrolase enzymes and which may permit a processive hydrolysis of the cellulose substrate. The close structural si milarity between the two enzymes implies that kinetics and chain-end specif icity experiments performed on the H. insolens enzyme are likely to be appl icable to the homologous T. reesei enzyme. These cast doubt on the descript ion of cellobiohydrolases as exoenzymes since they demonstrated that Cel6A (CBH II) shows no requirement for non-reducing chain-ends, as had been pres umed. There is no crystallographic evidence in the present structure to sup port a mechanism involving loop opening, yet preliminary modelling experime nts suggest that the active-site tunnel of Ce16A (CBH II) is too narrow to permit entry of a fluorescenylderivatize substrate, known to be a viable su bstrate for this enzyme.