Strategic aromatic residues in the catalytic cleft of the xyloglucanase MtXgh74 modifying thermostability, mode of enzyme action, and viscosity reduction ability.

The thermostable endo-processive xyloglucanase MtXgh74 from Myceliophthora thermophila was used to study the influence of aromatic amino acids in the catalytic cleft on the mode of action and the ability of enzyme to reduce xyloglucan viscosity. The enzyme derivative Mut I with mutations W64A/W67A in the "negative" subsites of the catalytic cleft resulted in a 5.5-fold increase of the Km value. Mut I produced oligosaccharides of various lengths in addition to xyloglucan building blocks. The W320A/W321A substitutions in the "positive" subsites of the mutated enzyme Mut II catalytic cleft increased the Km value 54-fold and resulted in an endo-dissociative mode of action. The ability of Mut II to reduce the viscosity of xyloglucan at 50 °C was much better than that of other MtXgh74 variants. Besides, Mut II efficiently reduced viscosity of a natural substrate, the pulp of xyloglucan-containing tamarind seed flour. The Km, Vmax, and kcat values and viscosity reduction ability of the enzyme derivative Mut III (W320A/W321A/G446Y) returned to levels close to that of MtXgh74. The pattern of xyloglucan hydrolysis by Mut III was typical for endo-processive xyloglucanases. The thermostability of Mut I and Mut II at 60 °C decreased significantly compared to the wild type, whereas the thermostability of Mut III at 60 °C restored almost to the MtXgh74-wt value. All mutants lost the ability to cleave the backbone of xyloglucan building blocks which was a characteristic of MtXgh74. Instead they acquired a low branch removing activity. Molecular dynamics simulations revealed the role of mutated amino acids in the complex action mechanism of GH74 enzymes. KEY POINTS: • Endo-processive mode of action of the xyloglucanase MtXgh74 was altered by rational design. • The endo-dissociative mutant Mut II (W320A/W321A) efficiently reduced XyG viscosity. • The substitutions W320A/W321A/G446Y in Mut III recovered the endo-processive mode. • Mut II can be used to reduce the viscosity of biomass slurries containing tamarind seed flour.