Protein engineering study of beta-mannosidase to set up a potential chemically efficient biocatalyst

0435519 - MBÚ 2015 RIV US eng J - Journal Article
Demo, G. - Horská, V. - Fliedrová, Barbora - Štěpán, J. - Koča, J. - Weignerová, Lenka - Křen, Vladimír - Wimmerová, M.
Protein engineering study of beta-mannosidase to set up a potential chemically efficient biocatalyst.
Glycobiology. Roč. 24, č. 12 (2014), s. 1301-1311. ISSN 0959-6658. E-ISSN 1460-2423
R&D Projects: GA ČR GAP207/10/0321; GA MŠMT(CZ) 7E11011
Grant - others:GA MŠk(CZ) LM2010005
Institutional support: RVO:61388971
Keywords : docking * molecular dynamics * mutagenesis
Subject RIV: CE - Biochemistry
Impact factor: 3.147, year: 2014

This study is focused on the analysis and mutagenesis of beta-mannosidase from Bacteroides thetaiotaomicron with the aim of broadening its substrate specificity to 2-acetamido-2-deoxy-beta-d-mannopyranosyl (beta-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 beta-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 beta-mannosidase's activity to include beta-ManNAc derivatives.
Permanent Link: http://hdl.handle.net/11104/0239457