Mutant barley (1-->3,1-->4)-beta-glucan endohydrolases with enhanced thermostability.
The similar three-dimensional structures of barley (1-->3)-beta-glucan endohydrolases and (1-->3,1-->4)-beta-glucan endohydrolases indicate that the enzymes are closely related in evolutionary terms. However, the (1-->3)-beta-glucanases hydrolyze polysaccharides of the type found in fungal cell walls and are members of the pathogenesis-related PR2 group of proteins, while the (1-->3,1-->4)-beta-glucanases function in plant cell wall metabolism. The (1-->3)-beta-glucanases have evolved to be significantly more stable 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, eight amino acid substitutions were introduced into a barley (1-->3,1-->4)-beta-glucanase using site-directed mutagenesis of a cDNA that encodes the enzyme. The amino acid substitutions chosen were based on structural comparisons of the barley (1-->3)- and (1-->3,1-->4)-beta-glucanases and of other higher plant (1-->3)-beta-glucanases. Three of the resulting mutant enzymes 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 mutation that was likely to decrease the entropy of the unfolded state of the enzyme. Furthermore, the three amino acid substitutions which increased the thermostability of barley (1-->3,1-->4)-beta-glucanase isoenzyme EII were all located in the COOH-terminal loop of the enzyme. Thus, this loop represents a particularly unstable region of the enzyme and could be involved in the initiation of unfolding of the (1-->3,1-->4)-beta-glucanase at elevated temperatures.
Stewart
R J
RJ
Department of Plant Science, University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Biomolecular Research Institute, 343 Royal Parade, Parkville, Vic 3052.
Varghese
J N
JN
Garrett
T P
TP
Høj
P B
PB
Fincher
G B
GB
D004355:Drug Stability; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics*; D001467:Hordeum / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D013696:Temperature; D013816:Thermodynamics
D006026:Glycoside Hydrolases; D004693:Glucan Endo-1,3-beta-D-Glucosidase; C028020:licheninase