Structural insights into the inhibition of cellobiohydrolases by xylo-oligosaccharides

One of the most important enzymes in industrial degradation and saccharification of cellulose are the cellobiohydrolases (CBHs) of CAZy Glucoside Hydrolase family 7. The most commonly used enzyme mixture component in such processes has been the CBH1 (also called Cel7A) from Trichoderma reesei (the anamorph to Hypocrea jecorina). However, the performance of the CBHs is susceptible to inhibition by compounds liberated by the physico-chemical pretreatment, especially if the biomass is kept unwashed. Xylan and xylooligo-saccharides (XOS) have been proposed to play a key role for this inhibition.

To shed light on the mechanism of inhibition at a molecular level, we have used X-ray crystallography to determine structures of T. reesei CBH1 in complex with XOS. Six different structure models of both the wild-type enzyme and catalytically deficient enzyme reveal a predominant binding mode at the beginning of the substrate-binding tunnel. This binding mode is slightly out of frame compared to the binding of cellooligosaccharides. Furthermore, a second, in-frame binding mode seems to be present, as evidenced by two consecutive xylose residues with partial occupancy in subsites -2 and -1 and thus in the vicinity of the catalytic center. Interestingly, the -1 xylosyl unit exhibits an open aldehyde conformation in one of the structures and a regular ring-closed pyranoside in another complex. Complementary inhibition studies with p-nitrophenyl lactoside as substrate indicate mixed inhibition rather than pure competitive inhibition, which may be explained by the two different binding modes