Epsilon Open Archive

Abstract

Glycoside hydrolase family 7 cellobiohydrolases (GH7 CBH) are typically the most abundant enzymes of cellulolytic fungi and play a key role in biomass recycling in Nature, as well as in biofuel production from plant biomass. This thesis examines molecular properties of this biologically and industrially important class of enzymes.

Paper I shows that HirCel7A is the most abundant protein of the serious forest pathogen Heterobasidion irregulare. The HirCel7A exhibits intermediate dynamical and structural properties between CBHs with the most closed and most open tunnels known in GH7. The results point to tunnel-enclosing loops as important for carbohydrate processivity and association-dissociation on cellulose. Paper II presents the first Michaelis complex, with cellononaose spanning 42 Å of the active site, and the first glycosyl-enzyme intermediate trapped in a GH7 CBH. QM/MM calculations determine optimal reaction coordinates, and rate constants at 11 s⁻¹ for Step1 and 5300 s⁻¹ for Step2, showing that the glycosylation step is rate-limiting. A product-assisted mechanism is revealed for the deglycosylation step, indicating that expulsion of the cellobiose product is not required prior to hydrolysis of the intermediate.

In Paper III, HgrCel7A from Humicola grisea var. thermoidea showed 10 °C higher Tm and 75% higher yield in a biomass performance assay at 65 °C than the canonical HjeCel7A of Hypocrea jecorina. The crystal structure of HgrCel7A indicates higher flexibility in tunnel-defining loops, and structural features potentially related to thermostability and enhanced activity, including a putative conformational switch in an active-center loop not reported previously in GH7. In Paper IV, structures of HjeCel7A in complex with xylooligosaccharides of DP 3-5 show predominant binding in the beginning of the tunnel and partial occupancy for a second binding mode near the catalytic centre. Birchwood xylan displayed ~100-fold stronger inhibition based on mass, suggesting that it may penetrate further into the tunnel and occupy a longer stretch of the active site.