title: Creating Space for Large Acceptors: Rational Biocatalyst Design for Resveratrol Glycosylation in an Aqueous System.
abstract: Polyphenols display a number of interesting properties but their low solubility limits practical applications. In that respect, glycosylation offers a solution for which sucrose phosphorylase has been proposed as a cost-effective biocatalyst. However, its activity on alternative acceptor substrates is too low for synthetic purposes and typically requires the addition of organic (co-)solvents. Here, we describe the engineering of the enzyme from Thermoanaerobacterium thermosaccharolyticum to enable glycosylation of resveratrol as test case. Based on docking and modeling studies, an active-site loop was predicted to hinder binding. Indeed, the unbolted loop variant R134A showed useful affinity for resveratrol (K(m)=185 mM) and could be used for the quantitative production of resveratrol 3-α-glucoside in an aqueous system. Improved activity was also shown for other acceptors, introducing variant R134A as promising new biocatalyst for glycosylation reactions on bulky phenolic acceptors.
authors.affiliation: Centre for Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, 9000 Ghent (Belgium).
authors.forename: Mareike E
authors.initials: ME
authors.lastname: Dirks-Hofmeister
authors.affiliation: Centre for Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, 9000 Ghent (Belgium).
authors.forename: Tom
authors.initials: T
authors.lastname: Verhaeghe
authors.affiliation: Centre for Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, 9000 Ghent (Belgium).
authors.forename: Karel
authors.initials: K
authors.lastname: De Winter
authors.affiliation: Centre for Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Coupure Links 653, 9000 Ghent (Belgium). tom.desmet@ugent.be.
authors.forename: Tom
authors.initials: T
authors.lastname: Desmet
mesh_terms: D001665:Binding Sites; D055162:Biocatalysis; D020134:Catalytic Domain; D004798:Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D000077185:Resveratrol; D013267:Stilbenes / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D046950:Thermoanaerobacterium / Q000201:enzymology; D014867:Water / Q000737:chemistry
chemical_list: D004798:Enzymes; D013267:Stilbenes; D014867:Water; D005964:Glucosyltransferases; C025901:sucrose phosphorylase; D000077185:Resveratrol
keywords: biocatalysis; glycosylation; protein engineering; resveratrol; sucrose phosphorylase