The amino acid on the top of the active groove allosterically modulates product specificity of the 1,4-α-glucan branching enzyme.

ABSTRACT

The 1,4-α-glucan branching enzymes (GBEs, EC 2.4.1.18) catalyse the formation of α-1,6 branching points in starch, presenting several potential applications in modifying starch. Previous study proved that W285 is considered to act as a "switch" to stop extension of substrates in the structure of GBE from Cyanothece sp. (cceBE). In the structure of GBE from Rhodothermus obamensis STB05 (RoGBE), the amino acid 160 site is structurally similar to the W285 in cceBE. In order to explore the role of this site in RoGBE, several engineered mutants individually substituted with Arg, Phe and Ala at G160 were studied in our research. The results show that substitution with Arg and Phe increased branching activity significantly, and the ratio of short glucan chains among all oligosaccharides increased. Finally, we proposed that the G160 is a 'door model' to elucidate introduced mutagenesis that triggers and controls the length of binding glucan chains of starch.