Lowering the pH optimum of D-xylose isomerase: the effect of mutations of the negatively charged residues.
Streptomyces rubiginosus D-xylose isomerase catalyzes the reversible isomerization of D-glucose to D-fructose. The isomerization reaction is maximized in the alkaline region of pH 8.5-8.8. The amino acid residues around two active site histidines (His-54 and His-220) and on the surface of the enzyme were mutated to improve the catalytic efficiency at neutral pH. The mutations have been made by removing the negatively charged residues based upon the sequence comparison of other D-xylose isomerases and the hypothesis proposed by Russell and Fersht (1987). The effects of these substitutions on kinetic parameter, pH dependence, and thermostability were characterized. The kcat values for D56N and E221A mutants on D-glucose are increased by 30-40% over that of the wild-type enzyme at pH 7.3 and the increased activities are maintained between pH 6 and 7.5. However, the surface mutants D65A, D81A, and D163N/E167Q only show 40-60% of the wild-type activity over the entire pH range. The pH activity profiles of the mutants are broader than that of the wild-type enzyme. The optimum pHs and the pKa values for all the mutants are lowered by 0.5-0.8 and 0.1-0.5 units, respectively. The small delta(deltaG) and high Tm values for all the mutants indicate that there is no significant change in the hydrogen bond network in the active site by mutations. These results indicate that D56N and E221A are possible candidates as good catalysts for High-Fructose Corn Syrup (HFCS) production.
Cha
J
J
Department of Food Science, Cornell University, Ithaca, New York 14853, USA. jaehocha@hotmail.com
Batt
C A
CA
D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D006639:Histidine / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012107:Research Design; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D013696:Temperature
D006639:Histidine; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase