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Probing impact of active site residue mutations on stability and activity of Neisseria polysaccharea amylosucrase
Authors:David Daudé  Christopher M. Topham  Magali Remaud‐Siméon  Isabelle André
Affiliation:1. Université de Toulouse;2. INSA, UPS,INP, LISBP, , F‐31077 Toulouse, France;3. CNRS, UMR5504, , F‐31400 Toulouse, France;4. INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, , F‐31400 Toulouse, France
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.
Keywords:amylosucrase  GH 13 family  enzyme engineering  enzyme thermostability  structure‐based approach  DSF
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