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 (Ala(224)) 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 A (=0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 A contact between N(epsilon2) and O(epsilon1). The pH variation of k(cat)/K(m) for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wild-type and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) 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 k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K(m) values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) 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.

pH profiles of cellulases depend on the substrate and architecture of the binding region

“…As argued above, pK a values obtained from measurements at very low pNPL concentrations may be taken as representative of the apoenzyme, and values in Table are in line with earlier suggestions based on the experimental (Becker et al, ) and computational (Bu, Crowley, Himmel, & Beckham, ) studies. For Cel7A, these earlier works assigned the low pK a value (pK a1 in Table ) to the nucleophile in the catalytic reaction (Glu212 in TrCel7A numbering), while the higher pK a (pK a2 ) was assigned to the catalytic acid/base, Glu217.…”

Sequencing, biochemical characterization, crystal structure and molecular dynamics of cellobiohydrolase Cel7A from Geotrichum candidum 3C

“…From a functional standpoint, the activity pH profile of Gca Cel7A is broader and slightly shifted in the alkaline direction, with an optimum at pH 5.0, as compared with 4.5 for Hje Cel7A . At pH 6 and pH 7, Gca Cel7A shows ~ 90% and ~ 60% activity, compared with ~ 75% and ~ 15% for Hje Cel7A.…”

Dissecting and Reconstructing Synergism

“…By contrast, hydrolysis rates of the natural insoluble substrate often decrease dramatically at later stages of hydrolysis (11–14). Therefore, many attempts have been made to enhance the intrinsic efficiency of cellulases (6, 15) and various types of cellulose pretreatment have been developed with the aim of maximizing substrate accessibility and reactivity toward enzymatic hydrolysis (6, 16). The actual source of this limitation, however, be it the enzymes, the substrate, or both, is a fundamentally unsolved puzzle (14).…”