Essential role of amino acid position 226 in oligosaccharide elongation by amylosucrase from Neisseria polysaccharea

• Published in: Biotechnology and bioengineering Vol. 111; no. 9; pp. 1719 - 1728
• Main Authors: Cambon, Emmanuelle, Barbe, Sophie, Pizzut-Serin, Sandra, Remaud-Simeon, Magali, André, Isabelle
• Format: Journal Article
• Language: English
• Published: United States Blackwell Publishing Ltd 01.09.2014; Wiley Subscription Services, Inc; Wiley
• Subjects: Amino Acid Substitution; Amino acids; Amino Acids - genetics; Amino Acids - metabolism; amylose synthesis; amylosucrase; Bioengineering; Catalysis; Catalysts; Catalytic Domain; Chain reactions; Chemical and Process Engineering; conformational rearrangements; DNA Mutational Analysis; Elongation; Engineering Sciences; Enzymes; Food engineering; GH13 family; Glucosyltransferases - chemistry; Glucosyltransferases - genetics; Glucosyltransferases - metabolism; Kinetics; Life Sciences; Models, Molecular; Mutagenesis, Site-Directed; Mutant Proteins - chemistry; Mutant Proteins - genetics; Mutant Proteins - metabolism; Neisseria - enzymology; Neisseria - genetics; Neisseria polysaccharea; Oligosaccharides - metabolism; Protein Conformation; rational engineering; Sucrose; Sucrose - metabolism; transglucosylase; amylosucrase; transglucosylase; rational engineering; GH13 family; conformational rearrangements; amylose synthesis
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.25236

ABSTRACT Amylosucrase from Neisseria polysaccharea is a remarkable transglucosylase that synthesizes an insoluble amylose‐like polymer from sole substrate sucrose. One particular amino acid, Arg226, was proposed from molecular modeling studies to play an important role in the formation of the active site topology and in the accessibility of ligands to the catalytic site. The systematic mutation of this Arg residue by all 19 other possible amino acids revealed that all single‐mutants had a higher activity on sucrose compared to the wild‐type enzyme. An extensive kinetic investigation showed that catalytic efficiencies are greatly impacted by the presence of natural acceptors in the reaction media, their chain length and the nature of the amino acid at position 226. Compared to the wild‐type enzyme, the R226N mutant showed a 10‐fold enhancement in the catalytic efficiency and a nearly twofold higher production of an insoluble amylose‐like polymer that can be of interest for biotechnological applications. Biotechnol. Bioeng. 2014;111: 1719–1728. © 2014 Wiley Periodicals, Inc. Neisseria polysaccharea amylosucrase catalyzes the production from sole sucrose of an insoluble amylose which can have many biotechnological applications. One particular amino acid residue, Arg226, was identified from molecular modeling studies to play a key role in substrate recognition and active site topology. Mutation of this arginine residue by other types of amino acids led to remarkably efficient enzymes on sucrose substrate and yielding remarkable levels of insoluble amylose compared to parental wild‐type amylosucrase.