Pullulanase, a typical debranching enzyme, specifically hydrolyzes α-1,6 glycosidic linkages in pullulan, starch and so on. There is accumulated knowledge about the catalysis of pullulanase, but functional roles of acceptor subsites get less attention. According to the crystal structure of pullulanase from Bacillus subtilis str. 168, stacking interaction between tryptophan437 and glycosyl unit of α-CD was found. Trp437 located in conserved region III was highly conserved in pullulanases. But functional role of this residue remains unclear. Site-directed mutagenesis was used to determine the function of Trp437. Replacement of Trp437 with glycine, phenylalanine, proline, arginine resulted in mutants with undetectable hydrolysis activity and reverse hydrolysis activity. The secondary structure of mutated proteins showed no difference from the wild-type pullulanase, but they lost the capability to bind β-CD based on the ITC measurement. Molecular docking was performed to investigate the affinity of proteins to ligands including maltotriose, 62-α-D-maltotriosyl-maltotriose and α-CD, showing the affinity of mutants to ligands became weaker compared to that of PulA. And the hydrogen bond between Trp437 and Glu525 in PulA only was found in mutant W437R. These results show that Trp437 at subsite +2 plays crucial role in catalysis of PulA, and it has little tolerance to change.
Keywords: Ligands recognition; Molecular docking; Pullulanase; Site-directed mutagenesis; Tryptophan437.
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