Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum: the pH behaviour of Trichoderma reesei Cel7A and its E223S/ A224H/L225V/T226A/D262G mutant

Abstract

The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala224) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves. The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 Å (= 0.1nm) closer to the acid/base Glu217 residue, with a 3.1 Å contact between N∊2 and O∊1. The pH variation of kcat/Km for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wild-type and mutant, with pK1 shifting from 2.22±0.03 in the wild-type to 3.19±0.03 in the mutant, and pK2 shifting from 5.99±0.02 to 6.78±0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative kcat for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower kcat/Km values for both lactosides and cellobiosides, and a marginally lower stability. However, kcat values for cellobiosides are higher for the mutant. This we attribute to reduced non-productive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.

Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases

“…The catalytic site is located at one end of the tunnel and enables product release from the reducing end of the cellulose chain, while the rest of the polymer chain is still bound to the enzyme. Structure-based protein engineering has been applied to study, for example, the details of the catalytic mechanism (Divne et al, 1998), the role of the surface loops and the pH behavior of Tr Cel7A (Becker et al, 2001;Ståhlberg et al, 1996).…”

Small Angle Neutron Scattering Reveals pH-dependent Conformational Changes in Trichoderma reesei Cellobiohydrolase I

“…reesei is an acidophilic organism typically cultivated at pH 4.8 or 5.0. Its cellulases function optimally at acidic pH between 3 and 5 (17,18). In particular, pH values between 4 and 5 are optimal for Cel7A (17,18).…”

The relationship between thermal stability and pH optimum studied with wild‐type and mutant Trichoderma reesei cellobiohydrolase Cel7A

“…Similar to many other cellulases, T. reesei Cel7A is composed of a large catalytic and a small cellulose-binding domain (CBD; now also called a carbohydrate-binding module, CBM), which are joined by an O-glycosylated linker peptide. Three-dimensional structures of both the isolated catalytic and cellulose-binding domain of wild-type and various mutated forms of Cel7A cellobiohydrolase have been solved [3,[4][5][6][7][8]. The catalytic domain of Cel7A has a large concave b-sandwich fold where individual b-strands are joined by long surface loops enclosing the active site in a tunnel, which spans through the whole catalytic domain.…”

“…One of the goals in cellulase engineering for these processes is to understand and alter the operative pH of these enzymes. Recently a variant of T. reesei Cel7A containing five point mutations (E223S/ A224H/L225V/T226A/D262G) and having a more alkaline pH optimum was produced [3]. The mutations were designed based on the sequence comparisons of family 7 cellulases having different pH behaviours.…”