Probing the role of helix α1 in the acid-tolerance and thermal stability of the Streptomyces sp. SK Glucose Isomerase by site-directed mutagenesis

In order to investigate the role of helix α1 in the different biochemical properties between class I and class II Glucose Isomerases, a histidine and a phenylalanine residue were inserted at position 17 and 19 of Streptomyces sp. SK Glucose Isomerase (SKGI). In addition, W16 was substituted by a histidine. The H17/F19 insertion displaced the optimal pH of SKGI from 6.5 to 7-8 and slightly decreased the thermostability. As for the W16H mutant, a shift in optimal pH of SKGI from 6.5 to 6 was observed along with a decrease in the enzyme thermostability at 85 °C with a half-life time reduced twice compared to the wild-type enzyme. Three-dimensional structure analysis suggested that the insertion of a histidine at position 17 results in the formation of new hydrogen bond with D287, thereby preventing it from deprotonating the O2 hydroxyl of the sugar at low pH, while the substitution W16H induced opposite effect by preventing hydrogen bond formation between D287 and W16 and thereby probably facilitating the hydrogen transfer during the isomerization reaction. The findings highlight the essential role of helix α1, which bears the three introduced mutations, in the acid-tolerance and the thermostability of SKGI and of glucose isomerases in general.