Mutant barley (1 -> 3,1 -> 4)-beta-glucan endohydrolases with enhanced thermostability

The similar three-dimensional structures of barley (1-->3)-beta -glucan end ohydrolases and (1-->3,1-->4)-beta -glucan endohydrolases indicate that the enzymes are closely related in evolutionary terms, However, the (1-->3)-be ta -glucanases hydrolyze polysaccharides of the type found in fungal cell w alls and are members of the pathogenesis-related PR2 group of proteins, whi le the (1-->3,1-->4)-beta -glucanases function in plant cell wall metabolis m, The (1-->3)-beta -glucanases have evolved to be significantly more stabl e than the (1-->3,1-->4)-beta -glucanases, probably as a consequence of the hostile environments imposed upon the plant by invading microorganisms. In attempts to define the molecular basis for the differences in stability, e ight amino acid substitutions were introduced into a barley (1-->3,1-->4)-b eta -glucanase using site-directed mutagenesis of a cDNA that encodes the e nzyme. The amino acid substitutions chosen were based on structural compari sons of the barley (1-->3)- and (-->3,1-->4)-beta -glucanases and of other higher plant (1-->3)-beta -glucanases. Three of the resulting mutant enzyme s showed increased thermostability compared with the wild-type (1-->3,1-->4 )-beta -glucanase. The largest increase in stability was observed when the histidine at position 300 was changed to a proline (mutant H300P), a mutati on that was likely to decrease the entropy of the unfolded state of the enz yme. Furthermore, the three amino acid substitutions which increased the th ermostability of barley (1-->3,1-->4)-beta -glucanase isoenzyme EII were al l located in the COOH-terminal loop of the enzyme. Thus, this loop represen ts a particularly unstable region of the enzyme and could be involved in th e initiation of unfolding of the (1-->3,1-->4)-beta -glucanase at elevated temperatures.