title: Protein engineering study of β-mannosidase to set up a potential chemically efficient biocatalyst.
abstract: This study is focused on the analysis and mutagenesis of β-mannosidase from Bacteroides thetaiotaomicron with the aim of broadening its substrate specificity to 2-acetamido-2-deoxy-β-d-mannopyranosyl (β-ManNAc) derivatives. Various conformations ((4)C1, (4)H5 and (1)S5) of native and modified ligands were docked to the binding site of the protein to determine the most suitable conformation of sugars for further hydrolysis. Key amino acid residues were mutated in silico focusing on stabilizing the acetamido group of β-ManNAc as well as forming the oxazoline intermediate needed for hydrolysis. The results of large set of 5 ns molecular dynamic simulations showed that the majority of the active site residues are involved in substrate interaction and do not exhibit a higher flexibility except for Asn178. Mutations of Asn178 to alanine and Asp199 to serine could lead to a stabilization of the acetamido group in the binding site. So far, in vitro mutagenesis and the screen of a large variety of biological sources were unable to extend β-mannosidase's activity to include β-ManNAc derivatives.
authors: National Centre for Biomolecular Research, Faculty of Science Central European Institute of Technology, Masaryk University, Kamenice 5/A4, Brno 62500, Czech Republic.
authors: Gabriel
authors: G
authors: Demo
authors: National Centre for Biomolecular Research, Faculty of Science.
authors: Veronika
authors: V
authors: Horská
authors: Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, Prague 142 20, Czech Republic.
authors: Barbora
authors: B
authors: Fliedrová
authors: National Centre for Biomolecular Research, Faculty of Science Central European Institute of Technology, Masaryk University, Kamenice 5/A4, Brno 62500, Czech Republic.
authors: Jakub
authors: J
authors: Stěpán
authors: National Centre for Biomolecular Research, Faculty of Science Central European Institute of Technology, Masaryk University, Kamenice 5/A4, Brno 62500, Czech Republic.
authors: Jaroslav
authors: J
authors: Koča
authors: Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, Prague 142 20, Czech Republic.
authors: Lenka
authors: L
authors: Weignerová
authors: Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, Prague 142 20, Czech Republic.
authors: Vladimír
authors: V
authors: Křen
authors: National Centre for Biomolecular Research, Faculty of Science Central European Institute of Technology, Masaryk University, Kamenice 5/A4, Brno 62500, Czech Republic Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5/A5, Brno 62500, Czech Republic michaw@chemi.muni.cz.
authors: Michaela
authors: M
authors: Wimmerová
mesh_terms: D001439:Bacteroides / Q000201:enzymology*; D055162:Biocatalysis; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D015202:Protein Engineering; D044902:beta-Mannosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D044902:beta-Mannosidase
keywords: docking; molecular dynamics; mutagenesis; protein engineering; β-mannosidase