Abstract: The glycoside hydrolase GH97 family is a unique family that contains both inverting and retaining members. It is unclear how the two catalytic mechanisms differentiate. The catalytic mechanism of PspAG97A, a member of GH97 family, was analyzed. It was proved that PspAG97A performs the inverting catalytic mechanism according to the ratio change of α-glucose and β-glucose produced by the enzyme at different reaction time detected through high performance liquid chromatography. We tried to change the inverting catalytic mechanism of PspAG97A to retaining by structural analysis and site-directed mutagenesis. The active site of PspAG97A was superimposed with that of BtGH97b belonging to the GH97 retaining subfamily, and the related sites G407, E377, E456 and chloride ion binding site R171 of PspAG97A were subjected to single point mutations and combined mutations to follow the catalytic device for the retaining type. The results showed that the activities of the mutants decreased significantly compared to the wild-type. We then analyzed the function of relevant residues. The above research not only deepens the understanding of the catalytic mechanism of PspAG97A, but also lays the foundation for the evolutionary relationship of two catalytic mechanisms within the GH97 family.

Key words: glycoside hydrolase family 97, catalytic mechanism, site-directed mutagenesis