title: Probing the active site of cellodextrin phosphorylase from Clostridium stercorarium: kinetic characterization, ligand docking, and site-directed mutagenesis.
abstract: Cellodextrin phosphorylase from Clostridium stercorarium has been recombinantly expressed in Escherichia coli for the first time. Kinetic characterization of the purified enzyme has revealed that aryl and alkyl β-glucosides can be efficiently glycosylated, an activity that has not yet been described for this enzyme class. To obtain a better understanding of the factors that determine the enzyme's specificity, homology modeling and ligand docking were applied. Residue W168 has been found to form a hydrophobic stacking interaction with the substrate in subsite +2, and its importance has been examined by means of site-directed mutagenesis. The mutant W168A retains about half of its catalytic activity, indicating that other residues also contribute to the binding affinity of subsite +2. Finally, residue D474 has been identified as the catalytic acid, interacting with the glycosidic oxygen between subsites -1 and +1. Mutating this residue results in complete loss of activity. These results, for the first time, provide an insight in the enzyme-substrate interactions that determine the activity and specificity of cellodextrin phosphorylases.
authors.affiliation: Department of Biochemical and Microbial Technology, Centre of Expertise-Industrial Biotechnology and Biocatalysis, Ghent University, 9000 Ghent, Belgium. haigiang.tran@ugent.be
authors.forename: Giang Hai
authors.initials: GH
authors.lastname: Tran
authors.forename: Tom
authors.initials: T
authors.lastname: Desmet
authors.forename: Manu R M
authors.initials: MR
authors.lastname: De Groeve
authors.forename: Wim
authors.initials: W
authors.lastname: Soetaert
mesh_terms: D020134:Catalytic Domain / Q000235:genetics; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D005964:Glucosyltransferases / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D008024:Ligands; D005964:Glucosyltransferases; C105024:cellodextrin phosphorylase