Prediction and structural studies of active
site of selected glycosyl hydrolases from Paenibacillus
thiaminolyticus
Katarína Hlat-Glembová, Eva Benešová, Zita Purkrtová, Vojtěch Spiwok
Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague, Technická 3, Prague 6, 166 28, Czech Republic
Glycosyl hydrolases β-D-galactosidase and α-L-fucosidase isolated from the bacterium Paenibacillus thiaminolyticus show a great potential because of their transglycosylation ability, which found a potential in synthesis of glycoconjugates. The aim of our research is to study active sites of these enzymes, as their crystal structures have not been solved yet. Our study is based on combination of theoretical and experimental methods. The structural model of each enzyme was predicted by using a homology modeling. According to the predicted structure of each enzyme, the catalytic residues were predicted. These residues were subjected to side direct mutagenesis to prove their catalytic role. Here we report the progress of mutagenesis experiments and kinetic characterization of mutants β-D-galactosidase_mut157, which was already been purified, β-D-galactosidase_mut233, which is now in the plasmid DNA state, α-L-fucosidase_mut186 and α-L-fucosidase_mut239, which are in the state of PCR products.