title: Probing impact of active site residue mutations on stability and activity of Neisseria polysaccharea amylosucrase.
abstract: The amylosucrase from Neisseria polysaccharea is a transglucosidase from the GH13 family of glycoside-hydrolases that naturally catalyzes the synthesis of α-glucans from the widely available donor sucrose. Interestingly, natural molecular evolution has modeled a dense hydrogen bond network at subsite -1 responsible for the specific recognition of sucrose and conversely, it has loosened interactions at the subsite +1 creating a highly promiscuous subsite +1. The residues forming these subsites are considered to be likely involved in the activity as well as the overall stability of the enzyme. To assess their role, a structure-based approach was followed to reshape the subsite -1. A strategy based on stability change predictions, using the FoldX algorithm, was considered to identify the best candidates for site-directed mutagenesis and guide the construction of a small targeted library. A miniaturized purification protocol was developed and both mutant stability and substrate promiscuity were explored. A range of 8 °C between extreme melting temperature values was observed and some variants were able to synthesize series of oligosaccharides with distributions differing from that of the parental enzyme. The crucial role of subsite -1 was thus highlighted and the biocatalysts generated can now be considered as starting points for further engineering purposes.
authors.affiliation: Université de Toulouse; INSA, UPS,INP, LISBP, 135 Avenue de Rangueil, F-31077, Toulouse, France; CNRS, UMR5504, F-31400, Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400, Toulouse, France.
authors.forename: David
authors.initials: D
authors.lastname: Daudé
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Topham
authors.forename: Magali
authors.initials: M
authors.lastname: Remaud-Siméon
authors.forename: Isabelle
authors.initials: I
authors.lastname: André
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D020134:Catalytic Domain / Q000235:genetics; D004795:Enzyme Stability; D019143:Evolution, Molecular; D014644:Genetic Variation; D005936:Glucans / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009343:Neisseria / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D005936:Glucans; D005964:Glucosyltransferases; C019700:amylosucrase
keywords: DSF; GH 13 family; amylosucrase; enzyme engineering; enzyme thermostability; structure-based approach